Methods and compositions for alteration of a cystic fibrosis transmembrane conductance regulator (cftr) gene

ABSTRACT

Nucleases and methods of using these nucleases for alteration of a CFTR gene and generation of cells and animal models.

CROSS-REFERENCE TO RELATED APPLICATIONS

The present application is a divisional application of U.S. patentapplication Ser. No. 13/558,101, filed Jul. 25, 2012, which claims thebenefit of U.S. Provisional Application No. 61/511,434 filed Jul. 25,2011, the disclosure of which is hereby incorporated by reference in itsentirety.

STATEMENT OF RIGHTS TO INVENTIONS MADE UNDER FEDERALLY SPONSOREDRESEARCH

This invention was made with government support under HL099559 awardedby The National Institutes of Health. The government may have certainrights in the invention.

TECHNICAL FIELD

The present disclosure is in the fields of genome editing.

BACKGROUND

Lung diseases, including inherited disorders such as Cystic Fibrosis(CF) and Surfactant Protein B (SP-B) Deficiency remain an issue inpediatric populations. SP-B deficiency is a rare lung disease whereprotein and fat molecules accumulate in the distant parts of the lungsand affect breathing. The disease is caused by a deficiency of the lungsurfactant protein B, primarily due to a defect in the SFTPB gene whichencodes the pulmonary-associated surfactant B protein (SPB), anamphipathic surfactant protein essential for lung function andhomeostasis after birth. The most common mutation in SP-B deficiency isa mutation designated “121ins2” which results in the nucleotide “C” atposition 131 being converted into “GAA.”

CF is an autosomal recessive disorder affecting 1 in 1500 to 4000 livebirths, and is one of the most common inherited pediatric disorders. Theprimary defect in CF is in the regulation of epithelial chloridetransport by a chloride channel protein encoded by the cystic fibrosistransmembrane conductance regulator (CFTR) gene. See, e.g., Kerem et al.(1989) Science 245:1073-1080; Kreda et al. (2005) Mol Biol Cell16:2154-2167. About 70% of mutations observed in CF patients result fromdeletion of three base pairs in CFTR's nucleotide sequence, resulting inthe loss of the amino acid phenylalanine located at position 508 in theprotein (a mutation referred to as ΔF508). In a wild type genome, aminoacid 507 is an isoleucine, and is encoded by the codon TAG where the Gis nucleotide 1652 in the gene Amino acid 508 is a phenylalanine,encoded by AAA. In the Δ508 mutation, the G from the 507 codon isdeleted along with the first two As of the 508 codon, such that themutation has the sequence TAA at the deleted 507-508 encoding position.TAA also encodes an isoleucine, but the phenylalanine at wild typeposition 508 is lost. For the ΔI507 deletion, either the isoleucine atposition 506 or 507 is deleted. For this mutation, the nucleotides at1648-1650 or 1651-1653 are lost, or some combination thereof to resultin only one isoleucine in the resultant protein. Compound (heterozygous)mutations (ΔF508 and ΔI507) have also been documented. See, e.g., Orozcoet al. (1994) Am J Med Genet. 51(2):137-9. CF patients, either compoundheterozygous ΔI507/ΔF508 or homozygous ΔF508/ΔF508, fail to express thefully glycosylated CFTR protein and the partially glycosylated proteinis not expressed on the cell surface (see, e.g., Kreda et al. (2005) MolBiol Cell 16:2154-2167; Cheng et al. (1990) Cell 63:827-834) as isrequired for CFTR function. Individuals bearing either the ΔI507 orΔF508 CFTR mutations at only one allele (i.e. wt/ΔI507 or wt/ΔF508) areCF carriers and exhibit no defects in lung cell function. See, e.g.,Kerem et al. (1990) Proc Natl Acad Sci USA 87:8447-8451.

Although several organ systems are affected by mutations in the CFTRgene, recurrent pulmonary infections are responsible for 80 to 90% ofthe deaths in CF patients. There is some controversy as to which humanlung cell types express CFTR, although recent data indicate that CFTRexpression is greatest in the proximal lung, and is predominantlyexpressed by ciliated cells present in surface airway epithelium. Kredaet al. (2005) Mol Biol Cell 16:2154-2167; Engelhardt et al. (1992) NatGenet 2:240-248; Engelhardt et al. (1994) J Clin Invest 93:737-749.

Attempts to treat CF via in vivo gene therapy have been hindered by theimmunogenic recognition and clearance of the viral vector used todeliver the CFTR transgene, failure to detect long-term expression ofCFTR, and likely an inability to achieve stable transduction of relevantstem/progenitor cell populations in the lung Mueller &Flotte (2008) ClinRev Allergy Immunol 35:164-178; Anson et al. (2006) Curr Gene Ther6:161-179. Recently there have been reports of the isolation of humanlung stem cells (see Kajstura et al., (2011) New England Journal ofMedicine 364(19):1795). The authors report that these cells could beisolated, maintained in culture and re-introduced into damaged mouselungs in vivo, where they were able to structurally integrate into thetissue and reform bronchioles, alveoli and pulmonary vessels.

Thus, there remains a need for the development of novel anti-CFstrategies, including treatments and model systems (in vitro such ascell lines and in vivo animal systems) to model and treat CF based oninvestigation of CFTR mutations and develop stem cells fortransplantation and treatment of pulmonary diseases.

SUMMARY

Disclosed herein are methods and compositions for altering a CFTR orSFTPB locus. Also described are models for studying the function of theCF gene (e.g., CFTR) or SFTPB (e.g., SP-B), models for CF and SP-Bdeficiency drug discovery and for treating CF or SP-B as well as methodsof making and using these model systems. The compositions and methodsdescribed herein can be used for genome editing of CFTR or SFTPB,including, but not limited to: cleaving of a CFTR or SFTPB gene in ananimal cell resulting in targeted alteration (insertion, deletion and/orsubstitution mutations) in the CFTR or SFTPB gene, including theincorporation of these targeted alterations into the germline; targetedintroduction into a CFTR or a SFTPB gene of non-endogenous nucleic acidsequences, the partial or complete inactivation of a CFTR gene in ananimal; correction of an SFTPB gene in an animal; methods of inducinghomology-directed repair at a CFTR or SFTPB locus; generation of apulmonary stem cell population with a corrected CFTR or SFTPB gene fortransplant into a patient in need thereof, and generation of transgenicanimals modified at a CFTR and/or SFTPB locus (e.g., rodents andnon-human primates).

In one aspect, described herein is a zinc-finger protein (ZFP) thatbinds to target site in a CFTR gene in a genome, wherein the ZFPcomprises one or more engineered zinc-finger binding domains. In oneembodiment, the ZFP is a zinc-finger nuclease (ZFN) that cleaves atarget genomic region of interest, wherein the ZFN comprises one or moreengineered zinc-finger binding domains and a nuclease cleavage domain orcleavage half-domain. Cleavage domains and cleavage half domains can beobtained, for example, from various restriction endonucleases and/orhoming endonucleases. In one embodiment, the cleavage half-domains arederived from a Type IIS restriction endonuclease (e.g., Fok I). Incertain embodiments, the zinc finger domain recognizes a target site ina CFTR gene. In some embodiments, the zinc finger domain recognizes atarget site in a mutated CFTR gene such that the ZFN pair will cleaveonly a mutated CFTR allele.

In one aspect, described herein is a zinc-finger protein (ZFP) thatbinds to target site in a SFTPB gene in a genome, wherein the ZFPcomprises one or more engineered zinc-finger binding domains. In oneembodiment, the ZFP is a zinc-finger nuclease (ZFN) that cleaves atarget genomic region of interest, wherein the ZFN comprises one or moreengineered zinc-finger binding domains and a nuclease cleavage domain orcleavage half-domain Cleavage domains and cleavage half domains can beobtained, for example, from various restriction endonucleases and/orhoming endonucleases. In one embodiment, the cleavage half-domains arederived from a Type IIS restriction endonuclease (e.g., Fok I). Incertain embodiments, the zinc finger domain recognizes a target site ina SFTPB gene.

The ZFN may bind to and/or cleave a CFTR or SFTPB gene within the codingregion of the gene or in a non-coding sequence within or adjacent to thegene, such as, for example, a leader sequence, trailer sequence orintron, or within a non-transcribed region, either upstream ordownstream of the coding region.

In another aspect, described herein is a TALE protein (Transcriptionactivator like) that binds to target site in a CFTR or SFTPB gene in agenome, wherein the TALE comprises one or more engineered TALE DNAbinding domains. In one embodiment, the TALE is a nuclease (TALEN) thatcleaves a target genomic region of interest, wherein the TALEN comprisesone or more engineered TALE DNA binding domains and a nuclease cleavagedomain or cleavage half-domain Cleavage domains and cleavage halfdomains can be obtained, for example, from various restrictionendonucleases and/or homing endonucleases. In one embodiment, thecleavage half-domains are derived from a Type IIS restrictionendonuclease (e.g. Fok I). In certain embodiments, the TALE DNA bindingdomain recognizes a target site in a CFTR or SFTPB gene.

The TALEN may bind to and/or cleave a CFTR or SFTPB gene within thecoding region of the gene or in a non-coding sequence within or adjacentto the gene, such as, for example, a leader sequence, trailer sequenceor intron, or within a non-transcribed region, either upstream ordownstream of the coding region. In certain embodiments, the TALE DNAbinding domain recognizes a target site in a CFTR gene. In someembodiments, the TALE DNA binding domain recognizes a target site in amutated CFTR gene such that the TALEN pair will cleave only a mutatedCFTR allele.

In another aspect, described herein are compositions comprising one ormore of the zinc-finger or TALE nucleases described herein. In certainembodiments, the composition comprises one or more zinc-finger or TALEnucleases in combination with a pharmaceutically acceptable excipient.

In another aspect, described herein is a polynucleotide encoding one ormore ZFNs or TALENs described herein. The polynucleotide may be, forexample, mRNA.

In another aspect, described herein is a ZFN or TALEN expression vectorcomprising a polynucleotide, encoding one or more ZFNs or TALENsdescribed herein, operably linked to a promoter.

In another aspect, described herein is a host cell comprising one ormore ZFN or TALEN expression vectors. The host cell may be stablytransformed or transiently transfected or a combination thereof with oneor more ZFP or TALEN expression vectors. In one embodiment, the hostcell is an embryonic stem cell. In one embodiment, the host cell is alung stem cell. In other embodiments, the one or more ZFP or TALENexpression vectors express one or more ZFNs or TALENs in the host cell.In another embodiment, the host cell may further comprise an exogenouspolynucleotide donor sequence. In any of the embodiments, describedherein, the host cell can be in an embryo, for example a one or moremouse, rat, rabbit or other mammal embryos (e.g., a non-human primate).

In another aspect, described herein is a method for cleaving one or moreCFTR or SFTPB genes in a cell, the method comprising: (a) introducing,into the cell, one or more polynucleotides encoding one or more ZFNs orTALENs that bind to a target site in the one or more genes underconditions such that the ZFN(s) is (are) or TALENs is (are) expressedand the one or more genes (CFTR and/or SFTPB) are cleaved.

In another embodiment, described herein is a method for modifying one ormore CFTR or SFTPB gene sequence(s) in the genome of a cell, the methodcomprising (a) providing a cell comprising one or more CFTR or SFTPBsequences; and (b) expressing first and second zinc-finger nucleases(ZFNs) or TALENs in the cell, wherein the first ZFN or TALEN cleaves ata first cleavage site and the second ZFN or TALEN cleaves at a secondcleavage site, wherein the gene sequence is located between the firstcleavage site and the second cleavage site, wherein cleavage of thefirst and second cleavage sites results in modification of the genesequence by non-homologous end joining and/or homology directed repair.Optionally, the cleavage results in insertion of an exogenous sequence(transgene) also introduced into the cell. In other embodiments,non-homologous end joining results in a deletion between the first andsecond cleavage sites. The size of the deletion in the gene sequence isdetermined by the distance between the first and second cleavage sites.Accordingly, deletions of any size, in any genomic region of interest,can be obtained. Deletions of 25, 50, 100, 200, 300, 400, 500, 600, 700,800, 900, 1,000 nucleotide pairs, or any integral value of nucleotidepairs within this range, can be obtained. In addition deletions of asequence of any integral value of nucleotide pairs greater than 1,000nucleotide pairs can be obtained using the methods and compositionsdisclosed herein. Using these methods and compositions, mutant CFTRand/or SFTPB proteins may be developed that lack one or more of theknown domains. These constructs can then be used to study the functionof the protein within a cell.

In another aspect, specific mutations associated with CFTR or SFTPB canbe corrected to understand the function of the gene that harbors themutation, and/or to discover phenotypes associated with the correctionof the mutant gene, including, for example, mutations ΔF508 and/or ΔI507in CFTR. Such an understanding then can be used to design cells, celllines and transgenic animals for use in drug screening and drugdiscovery, for example for treatments of CF or SP-B deficiency.

In another aspect, site specific mutations in CFTR or SFTPB can beconstructed to model known or novel mutations. For example, the ΔF508mutation in CFTR can be constructed in a cell, cell line, primary cellor transgenic animal. In one embodiment, a cell, cell line or transgenicanimal carrying a heterozygous genotype for CFTR is constructed, whilein another embodiment, a homozygous cell, cell line or transgenic animalis made carrying two mutant copies in both alleles of a desired locus.

In another aspect, described herein are methods of inactivating a CFTRor SFTPB gene in a cell by introducing one or more proteins,polynucleotides and/or vectors into the cell as described herein. In anyof the methods described herein the ZFNs or TALENs may induce targetedmutagenesis, targeted deletions of cellular DNA sequences, and/orfacilitate targeted recombination at a predetermined chromosomal locus.Thus, in certain embodiments, the ZFNs or TALENs delete or insert one ormore nucleotides of the target gene. In some embodiments, the CFTR orSFTPB gene is inactivated by ZFN or TALEN cleavage followed bynon-homologous end joining (NHEJ). In other embodiments, a genomicsequence in the target gene is replaced, for example using a ZFN orTALEN (or vector encoding said ZFN or TALEN) as described herein and a“donor” sequence that is inserted into the gene following targetedcleavage with the ZFN or TALEN. The donor sequence may be present in theZFN or TALEN vector, present in a separate vector (e.g., Ad or LVvector) or, alternatively, may be introduced into the cell using adifferent nucleic acid delivery mechanism. In one aspect, the donorsequence causes a known mutation, e.g., the ΔF508 mutation in the CFTRprotein. In certain embodiments, the donor sequence includes a sequencethat, following targeted integration of the donor sequence into a ΔF508mutant allele, results in a base pair substitution (A>G) in intron 9 ofCFTR (note, A>G substitution occurs at position −61 in intron 9 withrespect to start of exon 10:

i.e. —61A>G).

In another aspect, described herein are methods of correcting a CFTR orSFTPB gene (e.g., a mutant gene) in a cell by introducing one or moreproteins, polynucleotides and/or vectors into the cell as describedherein. In any of the methods described herein the ZFNs or TALENs mayinduce targeted mutagenesis, targeted deletions of cellular DNAsequences, and/or facilitate targeted recombination at a predeterminedchromosomal locus. Thus, in certain embodiments, the ZFNs or TALENsdelete or insert one or more nucleotides of or into the target gene. Insome embodiments the CFTR and/or SFTPB gene is corrected by ZFN or TALENcleavage followed by non-homologous end joining (NHEJ). In otherembodiments, a genomic sequence in the target gene is replaced, forexample using a ZFN or TALEN (or vector encoding said ZFN or TALEN) asdescribed herein and a “donor” sequence that is integrated into the genefollowing targeted cleavage with the ZFN or TALEN correcting thesequence of the CFTR or SFTPB gene. In some embodiments, the donorsequence is inserted into a safe harbor locus (see co-owned UnitedStates Patent publication 20080299580). The donor sequence may bepresent in the ZFN or TALEN vector, present in a separate vector (e.g.,Ad or LV vector) or, alternatively, may be introduced into the cellusing a different nucleic acid delivery mechanism. In one aspect, thedonor sequence corrects a known mutation, for example correction of theΔF508 mutation. In any of the embodiments described herein, thecorrection results in expression of a CFTR protein that is fullyglycosylated.

In any of the methods or compositions described herein, the cellcontaining the CFTR or SFTPB locus can be a stem cell. Specific stemcell types that may be used with the methods and compositions of theinvention include embryonic stem cells (ESC), hematopoietic stem cells,nerve stem cells, skin stem cells, muscle stem cells, lung stem cellsand induced pluripotent stem cells (iPSC). The iPSCs can be derived frompatient samples or from normal donors wherein the patient derived iPSCcan be mutated to normal gene sequence at the gene of interest, ornormal cells can be altered to the known disease allele at the gene ofinterest. Panels of these iPSC can be used to create isogenic cells withboth patient and normal cells carrying one or more mutations at theirendogenous CFTR or SFTPB loci. These cells can be used to create celllines and/or transgenic animals differing only at the mutations ofinterest to study multigene effects of disease severity and possibletherapeutic treatments for CF and/or SB-P deficiency. Other cell typesthat may be used for these studies are patient derived fibroblasts orpatient derived stem cells. In another aspect, the invention providesmethods and compositions for the development of lung (or other) stemcells for transplant into patients in need thereof. The lung stem cellsfor transplant may be derived from the patient, corrected at the diseaseassociated site in the CFTR or SFTPB locus and reintroduced into apatient. In other aspects the lung stem cells may be from a universalsource and contain a wild type CFTR or SFTPB gene, where the HLA and/orother self-markers have been altered (see co-owned United States PatentPublication No. 20120060230) such that the transplanted cells are notrejected by the patient.

In another aspect, described herein is a method of creating one or moreheritable mutant alleles in at least one CFTR or SFTPB locus ofinterest, the method comprising modifying one or more CFTR or SFTPB lociin the genome of one or more cells of an animal embryo by any of themethods described herein; raising the embryo to sexual maturity; andallowing the sexually mature animal to produce offspring; wherein atleast some of the offspring comprise the mutant alleles. In certainembodiments, the animal is a small mammal, for example a rabbit or arodent such as rat, a mouse or a guinea pig. In other embodiments, theanimal is a non-human primate.

In any of the methods described herein, the polynucleotide encoding thezinc finger nuclease(s) or TALEN(s) can comprise DNA, RNA orcombinations thereof. In certain embodiments, the polynucleotidecomprises a plasmid. In other embodiments, the polynucleotide encodingthe nuclease comprises mRNA.

In a still further aspect, provided herein is a method for site specificintegration of a nucleic acid sequence into a CFTR or SFTPB locus of achromosome. In certain embodiments, the method comprises: (a) injectingan embryo with (i) at least one DNA vector, wherein the DNA vectorcomprises an upstream sequence and a downstream sequence flanking thenucleic acid sequence to be integrated, and (ii) at least one RNAmolecule encoding a zinc finger or TALE nuclease that recognizes thesite of integration in the CFTR or SFTPB locus, and (b) culturing theembryo to allow expression of the zinc finger or TALE nuclease, whereina double stranded break introduced into the site of integration by thezinc finger nuclease or TALEN is repaired, via homologous recombinationwith the DNA vector, so as to integrate the nucleic acid sequence intothe chromosome.

Suitable embryos may be derived from several different vertebratespecies, including mammalian, bird, reptile, amphibian, and fishspecies. Generally speaking, a suitable embryo is an embryo that may becollected, injected, and cultured to allow the expression of a zincfinger or TALE nuclease. In some embodiments, suitable embryos mayinclude embryos from small mammals (e.g., rodents, rabbits, etc.),companion animals, livestock, or primates. Non-limiting examples ofrodents may include mice, rats, hamsters, gerbils, and guinea pigs.Non-limiting examples of companion animals may include cats, dogs,rabbits, hedgehogs, and ferrets. Non-limiting examples of livestock mayinclude horses, goats, sheep, swine, llamas, alpacas, and cattle.Non-limiting examples of primates may include capuchin monkeys,chimpanzees, lemurs, macaques, marmosets, tamarins, spider monkeys,squirrel monkeys, and vervet monkeys. In other embodiments, suitableembryos may include embryos from fish, reptiles, amphibians, or birds.Alternatively, suitable embryos may be insect embryos, for instance, aDrosophila embryo or a mosquito embryo.

Also provided is an embryo comprising at least one DNA vector, whereinthe DNA vector comprises an upstream sequence and a downstream sequenceflanking the nucleic acid sequence to be integrated, and at least oneRNA molecule encoding a zinc finger nuclease that recognizes thechromosomal site of integration. Organisms derived from any of theembryos as described herein are also provided.

In another aspect provided by the methods and compositions of theinvention is the use of cells, cell lines and animals (e.g., transgenicanimals) in the screening of drug libraries and/or other therapeuticcompositions (i.e., antibodies, structural RNAs, etc.) for use intreatment of an animal afflicted with CF or SB-P deficiency. Suchscreens can begin at the cellular level with manipulated cell lines orprimary cells, and can progress up to the level of treatment of a wholeanimal (e.g., human).

A kit, comprising the ZFPs or TALENs of the invention, is also provided.The kit may comprise nucleic acids encoding the ZFPs or TALENs, (e.g.RNA molecules or ZFP or TALEN encoding genes contained in a suitableexpression vector), or aliquots of the ZFP or TALEN proteins, donormolecules, suitable host cell lines, instructions for performing themethods of the invention, and the like.

These and other aspects will be readily apparent to the skilled artisanin light of this disclosure as a whole.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a schematic depicting targeted, ZFN or TALEN-mediatedcorrection of ΔI507 or ΔF508 CFTR mutations in the genomes of CF patientderived induced pluripotent stem cells (CF iPSC) and showing co-deliveryof CFTR-specific nuclease together with CFTR donor, followed byCre-recombinase mediated excision. lox: lox P sites; pgk: murinephosphoglycerate kinase promoter; puroTK: puromycin-thymidine kinasefusion gene; pA: polyadenylation signal sequences; Cre: Cre-recombinase.

FIG. 2, panels A to C, depict ZFN-mediated genome modification in CFiPSCs. FIG. 2A is a schematic depicting the targeted allele. Shown arethe primers 1/1′ utilized for upstream characterization (yielding a 1.8kb amplicon) and primers 2/2′ utilized for downstream characterization(yielding a 0.9 kb amplicon). FIG. 2B is a gel depicting identificationof an upstream 1.8 kb 1/1′ amplicon in 18 of 21 CFTR-targeted iPSCclones. iPSC clone numbers are shown above the individual lanes. Asshown, no 1.8 kb amplicon was identified for other puroR iPS clones(clones 17-22, 17-23), for the original CF clone 17 iPSC, nor for MEFs.FIG. 2C is a gel depicting identification of a downstream 0.9 kb 2/2′amplicon identified in 7 CFTR-targeted iPSC clones. As shown, no 0.9 kbamplicon was identified for 11 of the previously identified 18 targetedclones, for other puroR iPS clones (clones 17-22, 17-23), for theoriginal CF clone 17 iPSC, nor for MEFs.

FIG. 3, panels A and B, show expression of corrected CFTR mRNA byCFTR-edited iPSC clones. FIG. 3A is a gel showing RT-PCR analysis ofCFTR expression for the seven CFTR targeted CF iPS clones. Also shown isCFTR expression by the original Clone 17 CF ΔI507/ΔF508 iPS cells, WA09(H9) hES cells, and the A549 lung epithelial cell line. The expectedsize of PCR amplified cDNA (exons 8/9 to 11) is 0.46 kb. Analysis ofclones 17-1, 17-9, 17-14, and 17-16 yielded the expected band (indicatedon the Figure), whereas clones 17-13, 17-17, and 17-20 also exhibited alarger size band. FIG. 3B shows the sequence of CFTR RT-PCR product fromoriginal ΔI507/ΔF508 CFTR iPS cells (Clone 17, top strand showing ΔI507(SEQ ID NO:1) and bottom strand showing ΔF508 (SEQ ID NO:2)): correctedwt/ΔF508 CFTR iPS cells (Clone 17-1, top stand showing ΔF508 (SEQ IDNO:3), bottom strand showing wild type (wt) (SEQ ID NO:4)), and wt/wtCFTR A549 cells (SEQ ID NO:5).

FIG. 4, panels A and B, depict Cre-mediated excision of puroTK cassettefrom corrected CF wt/ΔF508 iPS cells. FIG. 4A is a schematic depictingthe modified allele before and after Cre-mediated excision, and theunmodified allele. The location of PCR primers 3 and 3′, both locatedoutside of donor sequences, used in verification by amplification areshown. Also indicated are the expected sizes of Cla I digestion productsfor modified and unmodified alleles. FIG. 4B is a gel showing RT-PCRanalysis of CFTR expression for two targeted CF iPS clones (17-9 and17-16) as well as their derived Cre-excised clones. Also shown is CFTRexpression by the original Clone 17 CF ΔI507/ΔF508 iPS cells, WA09 hEScells, and the A549 lung epithelial cell line. Sequencing of CFTR RT-PCRproduct from corrected wt/ΔF508 CFTR iPS cells (Clones 17-9 and 17-16),together with Cre-excised wt/ΔF508 CFTR iPS cells (17-9-C1 and 17-9-C2;17-16-C1 and 17-16-C2) is shown below the gel (top stand ΔF508 (SEQ IDNO:6) and bottom stand wt (SEQ ID NO:7)). Sequencing of the RT-PCRamplicon revealed equal mixture of wt and ΔF508 CFTR sequences in theCre-excised clones.

FIG. 5, panels A to C, show expression of corrected CFTR mRNA bycorrected CF iPS-derived cells. FIG. 5A shows gene expression patternsof original Clone 17 CF iPSC, either undifferentiated (d0) or followingculture in Activin A for 1-3 days showing clear up-regulation of bothSox17 and CFTR mRNAs over time. FIG. 5B shows gene expression pattern ofcorrected, Cre-excised Clone 17-9-C1 iPSC and also demonstratesup-regulation of both Sox17 and CFTR mRNA by days 3-5 of culture inActivin A. FIG. 5C shows gene expression levels in the indicated clonesand cells.

FIG. 6, panels A and B, depict additional CFTR-specific ZFNs. FIG. 6Ashows an illustration of the CFTR gene sequence showing the bindingsites for each of the ZFNs (SEQ ID NO:45 corresponds to the top DNAstrand, SEQ ID NO:46 is bottom DNA strand; SEQ ID NO:47 shows the aminoacid sequence; SEQ ID NO:48 shows a portion of the gene sequencecorresponding to the mutation, which is underlined in the Figure(TTATAGTAACCA)). The ZFNs bind sequences within Exon1 of CFTR.Additionally, a box is placed around the region where the Δ508 mutationcan arise. FIG. 6B depicts gels demonstrating ZFN-mediated genomemodification in K562 cells. The 32365/32366 and 32375/32376 ZFN pairscaused a 9% and 12% rate of indel formation, respectively.

DETAILED DESCRIPTION

Disclosed herein are compositions and methods for treating and/ordeveloping models useful in evaluating treatment of CF or SB-Pdeficiency. In particular, nuclease-mediated cleavage and integration isused to create or repair known mutations in the CFTR or SFTPB gene.These compositions and methods can be used to correct or create specificCFTR or SFTPB mutations in any selected genetic background to allow forstudy of CF or SB-P deficiency.

Thus, the methods and compositions described herein can be used tocreate isogenic panels of a set of mutations in CFTR or SFTPB to allowfor controlled study of these mutations, to investigate the link betweena certain mutation and cellular dysfunction and to identify phenotypesassociated with the mutation or with the correction of the mutation. Inaddition, any CFTR or SFTPB mutation can be introduced into patientderived cells, e.g. patient derived induced pluripotent stem cells(iPSCs), to investigate the effects of a certain mutation in a patientcell background. In addition, creation of CFTR or SFTPB mutants within-frame alterations is also part of the invention described herein, toallow for fine-tuned analysis of the functional domains of theseproteins. Furthermore, CFTR or SFTPB mutations associated with CF orSB-P2 can be created within the native gene in model animals (rat,non-human primate, etc.) to generate CF or SB-P deficiency models. Theseanimals may contain one or more inserted CFTR and/or SFTPB mutations.

Also described herein are methods and compositions for altering specificCFTR or SFTPB defects in patient cells. For example, mutated CFTR orSFTPB genes may be knocked out by use of specific nucleases that willonly act on mutant alleles and not act on a wild type gene sequence.Knock out of a specific gene may be a result of cleavage followed byNHEJ, or by cleavage at two loci within the gene to delete a largeportion of the gene, or by cleavage followed by targeted integration ofan oligonucleotide or larger donor DNA. Additionally, described hereinare methods and compositions to correct specific mutations in CFTR orSFTPB associated genes in patient cells. Such corrected cells may thenbe re-introduced back to the patient for treatment of CF or SF-Bdeficiency. Patient cells may be stem cells or iPSC. Universal stemcells may also be created using the methods of the invention which thenmay be used to treat any CF or SF-B patient.

General

Practice of the methods, as well as preparation and use of thecompositions disclosed herein employ, unless otherwise indicated,conventional techniques in molecular biology, biochemistry, chromatinstructure and analysis, computational chemistry, cell culture,recombinant DNA and related fields as are within the skill of the art.These techniques are fully explained in the literature. See, forexample, Sambrook et al. MOLECULAR CLONING: A LABORATORY MANUAL, Secondedition, Cold Spring Harbor Laboratory Press, 1989 and Third edition,2001; Ausubel et al., CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, John Wiley& Sons, New York, 1987 and periodic updates; the series METHODS INENZYMOLOGY, Academic Press, San Diego; Wolffe, CHROMATIN STRUCTURE ANDFUNCTION, Third edition, Academic Press, San Diego, 1998; METHODS INENZYMOLOGY, Vol. 304, “Chromatin” (P. M. Wassarman and A. P. Wolffe,eds.), Academic Press, San Diego, 1999; and METHODS IN MOLECULARBIOLOGY, Vol. 119, “Chromatin Protocols” (P. B. Becker, ed.) HumanaPress, Totowa, 1999.

DEFINITIONS

The terms “nucleic acid,” “polynucleotide,” and “oligonucleotide” areused interchangeably and refer to a deoxyribonucleotide orribonucleotide polymer, in linear or circular conformation, and ineither single- or double-stranded form. For the purposes of the presentdisclosure, these terms are not to be construed as limiting with respectto the length of a polymer. The terms can encompass known analogues ofnatural nucleotides, as well as nucleotides that are modified in thebase, sugar and/or phosphate moieties (e.g., phosphorothioatebackbones). In general, an analogue of a particular nucleotide has thesame base-pairing specificity; i.e., an analogue of A will base-pairwith T.

The terms “polypeptide,” “peptide” and “protein” are usedinterchangeably to refer to a polymer of amino acid residues. The termalso applies to amino acid polymers in which one or more amino acids arechemical analogues or modified derivatives of correspondingnaturally-occurring amino acids.

“Binding” refers to a sequence-specific, non-covalent interactionbetween macromolecules (e.g., between a protein and a nucleic acid). Notall components of a binding interaction need be sequence-specific (e.g.,contacts with phosphate residues in a DNA backbone), as long as theinteraction as a whole is sequence-specific. Such interactions aregenerally characterized by a dissociation constant (K_(d)) of 10⁻⁶ M⁻¹or lower. “Affinity” refers to the strength of binding: increasedbinding affinity being correlated with a lower K_(d).

A “binding protein” is a protein that is able to bind non-covalently toanother molecule. A binding protein can bind to, for example, a DNAmolecule (a DNA-binding protein), an RNA molecule (an RNA-bindingprotein) and/or a protein molecule (a protein-binding protein). In thecase of a protein-binding protein, it can bind to itself (to formhomodimers, homotrimers, etc.) and/or it can bind to one or moremolecules of a different protein or proteins. A binding protein can havemore than one type of binding activity. For example, zinc fingerproteins have DNA-binding, RNA-binding and protein-binding activity.

A “zinc finger DNA binding protein” (or binding domain) is a protein, ora domain within a larger protein, that binds DNA in a sequence-specificmanner through one or more zinc fingers, which are regions of amino acidsequence within the binding domain whose structure is stabilized throughcoordination of a zinc ion. The term zinc finger DNA binding protein isoften abbreviated as zinc finger protein or ZFP.

A “TALE DNA binding domain” or “TALE” is a polypeptide comprising one ormore TALE repeat domains/units. The repeat domains are involved inbinding of the TALE to its cognate target DNA sequence. A single “repeatunit” (also referred to as a “repeat”) is typically 33-35 amino acids inlength and exhibits at least some sequence homology with other TALErepeat sequences within a naturally occurring TALE protein.

Zinc finger binding domains can be “engineered” to bind to apredetermined nucleotide sequence, for example via engineering (alteringone or more amino acids) of the recognition helix region of a naturallyoccurring zinc finger protein. Similarly, TALEs can be “engineered” tobind to a predetermined nucleotide sequence, for example by engineeringof the amino acids involved in DNA binding (the RVD region). Therefore,engineered zinc finger proteins or TALE proteins are proteins that arenon-naturally occurring. Non-limiting examples of methods forengineering zinc finger proteins and TALEs are design and selection. Adesigned protein is a protein not occurring in nature whosedesign/composition results principally from rational criteria. Rationalcriteria for design include application of substitution rules andcomputerized algorithms for processing information in a database storinginformation of existing ZFP or TALE designs and binding data. See, forexample, U.S. Pat. Nos. 6,140,081; 6,453,242; and 6,534,261; see also WO98/53058; WO 98/53059; WO 98/53060; WO 02/016536 and WO 03/016496.

A “selected” zinc finger protein or TALE is a protein not found innature whose production results primarily from an empirical process suchas phage display, interaction trap or hybrid selection. See e.g., U.S.Pat. No. 5,789,538; U.S. Pat. No. 5,925,523; U.S. Pat. No. 6,007,988;U.S. Pat. No. 6,013,453; U.S. Pat. No. 6,200,759; WO 95/19431; WO96/06166; WO 98/53057; WO 98/54311; WO 00/27878; WO 01/60970 WO 01/88197and WO 02/099084.

“Recombination” refers to a process of exchange of genetic informationbetween two polynucleotides. For the purposes of this disclosure,“homologous recombination (HR)” refers to the specialized form of suchexchange that takes place, for example, during repair of double-strandbreaks in cells via homology-directed repair mechanisms. This processrequires nucleotide sequence homology, uses a “donor” molecule totemplate repair of a “target” molecule (i.e., the one that experiencedthe double-strand break), and is variously known as “non-crossover geneconversion” or “short tract gene conversion,” because it leads to thetransfer of genetic information from the donor to the target. Withoutwishing to be bound by any particular theory, such transfer can involvemismatch correction of heteroduplex DNA that forms between the brokentarget and the donor, and/or “synthesis-dependent strand annealing,” inwhich the donor is used to re-synthesize genetic information that willbecome part of the target, and/or related processes. Such specialized HRoften results in an alteration of the sequence of the target moleculesuch that part or all of the sequence of the donor polynucleotide isincorporated into the target polynucleotide.

In the methods of the disclosure, one or more targeted nucleases asdescribed herein create a double-stranded break in the target sequence(e.g., cellular chromatin) at a predetermined site, and a “donor”polynucleotide, having homology to the nucleotide sequence in the regionof the break, can be introduced into the cell. The presence of thedouble-stranded break has been shown to facilitate integration of thedonor sequence. The donor sequence may be physically integrated or,alternatively, the donor polynucleotide is used as a template for repairof the break via homologous recombination, resulting in the introductionof all or part of the nucleotide sequence as in the donor into thecellular chromatin. Thus, a first sequence in cellular chromatin can bealtered and, in certain embodiments, can be converted into a sequencepresent in a donor polynucleotide. Thus, the use of the terms “replace”or “replacement” can be understood to represent replacement of onenucleotide sequence by another, (i.e., replacement of a sequence in theinformational sense), and does not necessarily require physical orchemical replacement of one polynucleotide by another.

In any of the methods described herein, additional pairs of zinc-fingeror TALEN proteins can be used for additional double-stranded cleavage ofadditional target sites within the cell.

In certain embodiments of methods for targeted recombination and/orreplacement and/or alteration of a sequence in a region of interest incellular chromatin, a chromosomal sequence is altered by homologousrecombination with an exogenous “donor” nucleotide sequence. Suchhomologous recombination is stimulated by the presence of adouble-stranded break in cellular chromatin, if sequences homologous tothe region of the break are present.

In any of the methods described herein, the first nucleotide sequence(the “donor sequence”) can contain sequences that are homologous, butnot identical, to genomic sequences in the region of interest, therebystimulating homologous recombination to insert a non-identical sequencein the region of interest. Thus, in certain embodiments, portions of thedonor sequence that are homologous to sequences in the region ofinterest exhibit between about 80 to 99% (or any integer therebetween)sequence identity to the genomic sequence that is replaced. In otherembodiments, the homology between the donor and genomic sequence ishigher than 99%, for example if only 1 nucleotide differs as betweendonor and genomic sequences of over 100 contiguous base pairs. Incertain cases, a non-homologous portion of the donor sequence cancontain sequences not present in the region of interest, such that newsequences are introduced into the region of interest. In theseinstances, the non-homologous sequence is generally flanked by sequencesof 50-1,000 base pairs (or any integral value therebetween) or anynumber of base pairs greater than 1,000, that are homologous oridentical to sequences in the region of interest. In other embodiments,the donor sequence is non-homologous to the first sequence, and isinserted into the genome by non-homologous recombination mechanisms.

Any of the methods described herein can be used for partial or completeinactivation of one or more target sequences in a cell by targetedintegration of donor sequence that disrupts expression of the gene(s) ofinterest. Cell lines with partially or completely inactivated genes arealso provided.

Furthermore, the methods of targeted integration as described herein canalso be used to integrate one or more exogenous sequences. The exogenousnucleic acid sequence can comprise, for example, one or more genes orcDNA molecules, or any type of coding or non-coding sequence, as well asone or more control elements (e.g., promoters). In addition, theexogenous nucleic acid sequence may produce one or more RNA molecules(e.g., small hairpin RNAs (shRNAs), inhibitory RNAs (RNAis), microRNAs(miRNAs), etc.).

“Cleavage” refers to the breakage of the covalent backbone of a DNAmolecule. Cleavage can be initiated by a variety of methods including,but not limited to, enzymatic or chemical hydrolysis of a phosphodiesterbond. Both single-stranded cleavage and double-stranded cleavage arepossible, and double-stranded cleavage can occur as a result of twodistinct single-stranded cleavage events. DNA cleavage can result in theproduction of either blunt ends or staggered ends. In certainembodiments, fusion polypeptides are used for targeted double-strandedDNA cleavage.

A “cleavage half-domain” is a polypeptide sequence which, in conjunctionwith a second polypeptide (either identical or different) forms acomplex having cleavage activity (preferably double-strand cleavageactivity). The terms “first and second cleavage half-domains;” “+ and −cleavage half-domains” and “right and left cleavage half-domains” areused interchangeably to refer to pairs of cleavage half-domains thatdimerize.

An “engineered cleavage half-domain” is a cleavage half-domain that hasbeen modified so as to form obligate heterodimers with another cleavagehalf-domain (e.g., another engineered cleavage half-domain). See, also,U.S. Patent Publication Nos. 2005/0064474, 20070218528 and 2008/0131962,incorporated herein by reference in their entireties.

The term “sequence” refers to a nucleotide sequence of any length, whichcan be DNA or RNA; can be linear, circular or branched and can be eithersingle-stranded or double stranded. The term “donor sequence” refers toa nucleotide sequence that is inserted into a genome. A donor sequencecan be of any length, for example between 2 and 10,000 nucleotides inlength (or any integer value therebetween or thereabove), preferablybetween about 100 and 1,000 nucleotides in length (or any integertherebetween), more preferably between about 200 and 500 nucleotides inlength.

“Chromatin” is the nucleoprotein structure comprising the cellulargenome. Cellular chromatin comprises nucleic acid, primarily DNA, andprotein, including histones and non-histone chromosomal proteins. Themajority of eukaryotic cellular chromatin exists in the form ofnucleosomes, wherein a nucleosome core comprises approximately 150 basepairs of DNA associated with an octamer comprising two each of histonesH2A, H2B, H3 and H4; and linker DNA (of variable length depending on theorganism) extends between nucleosome cores. A molecule of histone H1 isgenerally associated with the linker DNA. For the purposes of thepresent disclosure, the term “chromatin” is meant to encompass all typesof cellular nucleoprotein, both prokaryotic and eukaryotic. Cellularchromatin includes both chromosomal and episomal chromatin.

A “chromosome,” is a chromatin complex comprising all or a portion ofthe genome of a cell. The genome of a cell is often characterized by itskaryotype, which is the collection of all the chromosomes that comprisethe genome of the cell. The genome of a cell can comprise one or morechromosomes.

An “episome” is a replicating nucleic acid, nucleoprotein complex orother structure comprising a nucleic acid that is not part of thechromosomal karyotype of a cell. Examples of episomes include plasmidsand certain viral genomes.

A “target site” or “target sequence” is a nucleic acid sequence thatdefines a portion of a nucleic acid to which a binding molecule willbind, provided sufficient conditions for binding exist.

An “exogenous” molecule is a molecule that is not normally present in acell, but can be introduced into a cell by one or more genetic,biochemical or other methods. “Normal presence in the cell” isdetermined with respect to the particular developmental stage andenvironmental conditions of the cell. Thus, for example, a molecule thatis present only during embryonic development of muscle is an exogenousmolecule with respect to an adult muscle cell. Similarly, a moleculeinduced by heat shock is an exogenous molecule with respect to anon-heat-shocked cell. An exogenous molecule can comprise, for example,a functioning version of a malfunctioning endogenous molecule or amalfunctioning version of a normally-functioning endogenous molecule.

An exogenous molecule can be, among other things, a small molecule, suchas is generated by a combinatorial chemistry process, or a macromoleculesuch as a protein, nucleic acid, carbohydrate, lipid, glycoprotein,lipoprotein, polysaccharide, any modified derivative of the abovemolecules, or any complex comprising one or more of the above molecules.Nucleic acids include DNA and RNA, can be single- or double-stranded;can be linear, branched or circular; and can be of any length. Nucleicacids include those capable of forming duplexes, as well astriplex-forming nucleic acids. See, for example, U.S. Pat. Nos.5,176,996 and 5,422,251. Proteins include, but are not limited to,DNA-binding proteins, transcription factors, chromatin remodelingfactors, methylated DNA binding proteins, polymerases, methylases,demethylases, acetylases, deacetylases, kinases, phosphatases,integrases, recombinases, ligases, topoisomerases, gyrases andhelicases.

An exogenous molecule can be the same type of molecule as an endogenousmolecule, e.g., an exogenous protein or nucleic acid. For example, anexogenous nucleic acid can comprise an infecting viral genome, a plasmidor episome introduced into a cell, or a chromosome that is not normallypresent in the cell. Methods for the introduction of exogenous moleculesinto cells are known to those of skill in the art and include, but arenot limited to, lipid-mediated transfer (i.e., liposomes, includingneutral and cationic lipids), electroporation, direct injection, cellfusion, particle bombardment, calcium phosphate co-precipitation,DEAE-dextran-mediated transfer and viral vector-mediated transfer. Anexogenous molecule can also be the same type of molecule as anendogenous molecule but derived from a different species than the cellis derived from. For example, a human nucleic acid sequence may beintroduced into a cell line originally derived from a mouse or hamster.

By contrast, an “endogenous” molecule is one that is normally present ina particular cell at a particular developmental stage under particularenvironmental conditions. For example, an endogenous nucleic acid cancomprise a chromosome, the genome of a mitochondrion, chloroplast orother organelle, or a naturally-occurring episomal nucleic acid.Additional endogenous molecules can include proteins, for example,transcription factors and enzymes.

A “fusion” molecule is a molecule in which two or more subunit moleculesare linked, preferably covalently. The subunit molecules can be the samechemical type of molecule, or can be different chemical types ofmolecules. Examples of the first type of fusion molecule include, butare not limited to, fusion proteins (for example, a fusion between a ZFPor TALE DNA-binding domain and one or more activation domains) andfusion nucleic acids (for example, a nucleic acid encoding the fusionprotein described supra). Examples of the second type of fusion moleculeinclude, but are not limited to, a fusion between a triplex-formingnucleic acid and a polypeptide, and a fusion between a minor groovebinder and a nucleic acid.

Expression of a fusion protein in a cell can result from delivery of thefusion protein to the cell or by delivery of a polynucleotide encodingthe fusion protein to a cell, wherein the polynucleotide is transcribed,and the transcript is translated, to generate the fusion protein.Trans-splicing, polypeptide cleavage and polypeptide ligation can alsobe involved in expression of a protein in a cell. Methods forpolynucleotide and polypeptide delivery to cells are presented elsewherein this disclosure.

A “gene,” for the purposes of the present disclosure, includes a DNAregion encoding a gene product (see infra), as well as all DNA regionswhich regulate the production of the gene product, whether or not suchregulatory sequences are adjacent to coding and/or transcribedsequences. Accordingly, a gene includes, but is not necessarily limitedto, promoter sequences, terminators, translational regulatory sequencessuch as ribosome binding sites and internal ribosome entry sites,enhancers, silencers, insulators, boundary elements, replicationorigins, matrix attachment sites and locus control regions.

“Gene expression” refers to the conversion of the information, containedin a gene, into a gene product. A gene product can be the directtranscriptional product of a gene (e.g., mRNA, tRNA, rRNA, antisenseRNA, ribozyme, structural RNA or any other type of RNA) or a proteinproduced by translation of an mRNA. Gene products also include RNAswhich are modified, by processes such as capping, polyadenylation,methylation, and editing, and proteins modified by, for example,methylation, acetylation, phosphorylation, ubiquitination,ADP-ribosylation, myristilation, and glycosylation.

“Modulation” of gene expression refers to a change in the activity of agene. Modulation of expression can include, but is not limited to, geneactivation and gene repression. Genome editing (e.g., cleavage,alteration, inactivation, random mutation) can be used to modulateexpression. Gene inactivation refers to any reduction in gene expressionas compared to a cell that does not include a ZFP or TALEN as describedherein. Thus, gene inactivation may be partial or complete.

A “region of interest” is any region of cellular chromatin, such as, forexample, a gene or a non-coding sequence within or adjacent to a gene,in which it is desirable to bind an exogenous molecule. Binding can befor the purposes of targeted DNA cleavage and/or targeted recombination.A region of interest can be present in a chromosome, an episome, anorganellar genome (e.g., mitochondrial, chloroplast), or an infectingviral genome, for example. A region of interest can be within the codingregion of a gene, within transcribed non-coding regions such as, forexample, leader sequences, trailer sequences or introns, or withinnon-transcribed regions, either upstream or downstream of the codingregion. A region of interest can be as small as a single nucleotide pairor up to 2,000 nucleotide pairs in length, or any integral value ofnucleotide pairs.

“Eukaryotic” cells include, but are not limited to, fungal cells (suchas yeast), plant cells, animal cells, mammalian cells and human cells(e.g., T-cells).

The terms “operative linkage” and “operatively linked” (or “operablylinked”) are used interchangeably with reference to a juxtaposition oftwo or more components (such as sequence elements), in which thecomponents are arranged such that both components function normally andallow the possibility that at least one of the components can mediate afunction that is exerted upon at least one of the other components. Byway of illustration, a transcriptional regulatory sequence, such as apromoter, is operatively linked to a coding sequence if thetranscriptional regulatory sequence controls the level of transcriptionof the coding sequence in response to the presence or absence of one ormore transcriptional regulatory factors. A transcriptional regulatorysequence is generally operatively linked in cis with a coding sequence,but need not be directly adjacent to it. For example, an enhancer is atranscriptional regulatory sequence that is operatively linked to acoding sequence, even though they are not contiguous.

With respect to fusion polypeptides, the term “operatively linked” canrefer to the fact that each of the components performs the same functionin linkage to the other component as it would if it were not so linked.For example, with respect to a fusion polypeptide in which a ZFP or TALEDNA-binding domain is fused to an activation domain, the ZFP or TALEDNA-binding domain and the activation domain are in operative linkageif, in the fusion polypeptide, the ZFP or TALE DNA-binding domainportion is able to bind its target site and/or its binding site, whilethe activation domain is able to up-regulate gene expression. When afusion polypeptide in which a ZFP or TALE DNA-binding domain is fused toa cleavage domain, the ZFP or TALE DNA-binding domain and the cleavagedomain are in operative linkage if, in the fusion polypeptide, the ZFPor TALE DNA-binding domain portion is able to bind its target siteand/or its binding site, while the cleavage domain is able to cleave DNAin the vicinity of the target site.

A “functional fragment” of a protein, polypeptide or nucleic acid is aprotein, polypeptide or nucleic acid whose sequence is not identical tothe full-length protein, polypeptide or nucleic acid, yet retains thesame function as the full-length protein, polypeptide or nucleic acid. Afunctional fragment can possess more, fewer, or the same number ofresidues as the corresponding native molecule, and/or can contain one ormore amino acid or nucleotide substitutions. Methods for determining thefunction of a nucleic acid (e.g., coding function, ability to hybridizeto another nucleic acid) are well-known in the art. Similarly, methodsfor determining protein function are well-known. For example, theDNA-binding function of a polypeptide can be determined, for example, byfilter-binding, electrophoretic mobility-shift, or immunoprecipitationassays. DNA cleavage can be assayed by gel electrophoresis. See Ausubelet al., supra. The ability of a protein to interact with another proteincan be determined, for example, by co-immunoprecipitation, two-hybridassays or complementation, both genetic and biochemical. See, forexample, Fields et al. (1989) Nature 340:245-246; U.S. Pat. No.5,585,245 and PCT WO 98/44350.

A “vector” is capable of transferring gene sequences to target cells.Typically, “vector construct,” “expression vector,” and “gene transfervector,” mean any nucleic acid construct capable of directing theexpression of a gene of interest and which can transfer gene sequencesto target cells. Thus, the term includes cloning, and expressionvehicles, as well as integrating vectors.

A “reporter gene” or “reporter sequence” refers to any sequence thatproduces a protein product that is easily measured, preferably althoughnot necessarily in a routine assay. Suitable reporter genes include, butare not limited to, sequences encoding proteins that mediate antibioticresistance (e.g., ampicillin resistance, neomycin resistance, G418resistance, puromycin resistance), sequences encoding colored orfluorescent or luminescent proteins (e.g., green fluorescent protein,enhanced green fluorescent protein, red fluorescent protein,luciferase), and proteins which mediate enhanced cell growth and/or geneamplification (e.g., dihydrofolatereductase). Epitope tags include, forexample, one or more copies of FLAG, His, myc, Tap, HA or any detectableamino acid sequence. “Expression tags” include sequences that encodereporters that may be operably linked to a desired gene sequence inorder to monitor expression of the gene of interest.

Nucleases

Described herein are compositions, particularly nucleases, which areuseful in correction of one or more mutant CFTR alleles and/or mutationof one or more CFTR alleles, for example to generate models of CF. Incertain embodiments, the nuclease is naturally occurring. In otherembodiments, the nuclease is non-naturally occurring, i.e., engineeredin the DNA-binding domain and/or cleavage domain. For example, theDNA-binding domain of a naturally-occurring nuclease may be altered tobind to a selected target site (e.g., a meganuclease that has beenengineered to bind to site different than the cognate binding site). Inother embodiments, the nuclease comprises heterologous DNA-binding andcleavage domains (e.g., zinc finger nucleases; TAL-effector nucleases;meganuclease DNA-binding domains with heterologous cleavage domains).

A. DNA-Binding Domains

In certain embodiments, the nuclease is a meganuclease (homingendonuclease). Naturally-occurring meganucleases recognize 15-40base-pair cleavage sites and are commonly grouped into four families:the LAGLIDADG (SEQ ID NO: 49) family, the GIY-YIG family, the His-Cystbox family and the HNH family. Exemplary homing endonucleases includeI-SceI, I-CeuI, PI-PspI, PI-Sce, I-SceIV, I-CsmI, I-PanI, I-SceII,I-PpoI, I-SceIII, I-CreI, I-TevI, I-TevII and I-TevIII. Theirrecognition sequences are known. See also U.S. Pat. No. 5,420,032; U.S.Pat. No. 6,833,252; Belfort et al. (1997) Nucleic Acids Res. 25:3379-3388; Dujon et al. (1989) Gene 82:115-118; Perler et al. (1994)Nucleic Acids Res. 22, 1125-1127; Jasin (1996) Trends Genet. 12:224-228;Gimble et al. (1996)J Mol. Biol. 263:163-180; Argast et al. (1998) JMol. Biol. 280:345-353 and the New England Biolabs catalogue.

In certain embodiments, the nuclease comprises an engineered(non-naturally occurring) homing endonuclease (meganuclease). Therecognition sequences of homing endonucleases and meganucleases such asI-SceI, I-CeuI, PI-PspI, PI-Sce, I-SceIV, I-CsmI, I-PanI, I-SceII,I-PpoI, I-SceIII, I-CreI, I-TevI, I-TevII and I-TevIII are known. Seealso U.S. Pat. No. 5,420,032; U.S. Pat. No. 6,833,252; Belfort et al.(1997) Nucleic Acids Res. 25:3379-3388; Dujon et al. (1989) Gene82:115-118; Perler et al. (1994) Nucleic Acids Res. 22, 1125-1127; Jasin(1996) Trends Genet. 12:224-228; Gimble et al. (1996) J. Mol. Biol.263:163-180; Argast et al. (1998) J. Mol. Biol. 280:345-353 and the NewEngland Biolabs catalogue. In addition, the DNA-binding specificity ofhoming endonucleases and meganucleases can be engineered to bindnon-natural target sites. See, for example, Chevalier et al. (2002)Molec. Cell 10:895-905; Epinat et al. (2003) Nucleic Acids Res.31:2952-2962; Ashworth et al. (2006) Nature 441:656-659; Paques et al.(2007) Current Gene Therapy 7:49-66; U.S. Patent Publication No.20070117128. The DNA-binding domains of the homing endonucleases andmeganucleases may be altered in the context of the nuclease as a whole(i.e., such that the nuclease includes the cognate cleavage domain) ormay be fused to a heterologous cleavage domain.

In other embodiments, the DNA-binding domain comprises a naturallyoccurring or engineered (non-naturally occurring) TAL effector DNAbinding domain. See, e.g., U.S. Patent Publication No. 20110301073,incorporated by reference in its entirety herein. The plant pathogenicbacteria of the genus Xanthomonas are known to cause many diseases inimportant crop plants. Pathogenicity of Xanthomonas depends on aconserved type III secretion (T3S) system which injects more than 25different effector proteins into the plant cell. Among these injectedproteins are transcription activator-like effectors (TALE) which mimicplant transcriptional activators and manipulate the plant transcriptome(see Kay et al (2007) Science 318:648-651). These proteins contain a DNAbinding domain and a transcriptional activation domain. One of the mostwell characterized TALEs is AvrBs3 from Xanthomonas campestris pv.Vesicatoria (see Bonas et al (1989) Mol Gen Genet 218: 127-136 andWO2010079430). TALEs contain a centralized domain of tandem repeats,each repeat containing approximately 34 amino acids, which are key tothe DNA binding specificity of these proteins. In addition, they containa nuclear localization sequence and an acidic transcriptional activationdomain (for a review see Schornack S, et al (2006) J Plant Physiol163(3): 256-272). In addition, in the phytopathogenic bacteria Ralstoniasolanacearum two genes, designated brg11 and hpx17 have been found thatare homologous to the AvrBs3 family of Xanthomonas in the R.solanacearum biovar 1 strain GMI1000 and in the biovar 4 strain RS1000(See Heuer et al (2007) Appl and Envir Micro 73(13): 4379-4384). Thesegenes are 98.9% identical in nucleotide sequence to each other butdiffer by a deletion of 1,575 bp in the repeat domain of hpx17. However,both gene products have less than 40% sequence identity with AvrBs3family proteins of Xanthomonas.

Thus, in some embodiments, the DNA binding domain that binds to a targetsite a CFTR gene is an engineered domain from a TAL effector similar tothose derived from the plant pathogens Xanthomonas (see Boch et al,(2009) Science 326: 1509-1512 and Moscou and Bogdanove, (2009) Science326: 1501) and Ralstonia (see Heuer et al (2007) Applied andEnvironmental Microbiology 73(13): 4379-4384); U.S. Patent PublicationNos. 20110301073 and 20110145940.

In certain embodiments, the DNA binding domain that binds to a targetsite a CFTR gene comprises a zinc finger protein. Preferably, the zincfinger protein is non-naturally occurring in that it is engineered tobind to a target site of choice. See, for example, See, for example,Beerli et al. (2002) Nature Biotechnol. 20:135-141; Pabo et al. (2001)Ann. Rev. Biochem. 70:313-340; Isalan et al. (2001) Nature Biotechnol.19:656-660; Segal et al. (2001) Curr. Opin. Biotechnol. 12:632-637; Chooet al. (2000) Curr. Opin. Struct. Biol. 10:411-416; U.S. Pat. Nos.6,453,242; 6,534,261; 6,599,692; 6,503,717; 6,689,558; 7,030,215;6,794,136; 7,067,317; 7,262,054; 7,070,934; 7,361,635; 7,253,273; andU.S. Patent Publication Nos. 2005/0064474; 2007/0218528; 2005/0267061,all incorporated herein by reference in their entireties.

An engineered zinc finger binding domain can have a novel bindingspecificity, compared to a naturally-occurring zinc finger protein.Engineering methods include, but are not limited to, rational design andvarious types of selection. Rational design includes, for example, usingdatabases comprising triplet (or quadruplet) nucleotide sequences andindividual zinc finger amino acid sequences, in which each triplet orquadruplet nucleotide sequence is associated with one or more amino acidsequences of zinc fingers which bind the particular triplet orquadruplet sequence. See, for example, co-owned U.S. Pat. Nos. 6,453,242and 6,534,261, incorporated by reference herein in their entireties.

Exemplary selection methods, including phage display and two-hybridsystems, are disclosed in U.S. Pat. Nos. 5,789,538; 5,925,523;6,007,988; 6,013,453; 6,410,248; 6,140,466; 6,200,759; and 6,242,568; aswell as WO 98/37186; WO 98/53057; WO 00/27878; WO 01/88197 and GB2,338,237. In addition, enhancement of binding specificity for zincfinger binding domains has been described, for example, in co-owned WO02/077227.

In addition, as disclosed in these and other references, DNA domains(e.g., multi-fingered zinc finger proteins) may be linked together usingany suitable linker sequences, including for example, linkers of 5 ormore amino acids in length. See, also, U.S. Pat. Nos. 6,479,626;6,903,185; and 7,153,949 for exemplary linker sequences 6 or more aminoacids in length. The zinc finger proteins described herein may includeany combination of suitable linkers between the individual zinc fingersof the protein. In addition, enhancement of binding specificity for zincfinger binding domains has been described, for example, in co-owned WO02/077227.

Selection of target sites; DNA-binding domains and methods for designand construction of fusion proteins (and polynucleotides encoding same)are known to those of skill in the art and described in detail in U.S.Pat. Nos. 6,140,081; 5,789,538; 6,453,242; 6,534,261; 5,925,523;6,007,988; 6,013,453; 6,200,759; WO 95/19431; WO 96/06166; WO 98/53057;WO 98/54311; WO 00/27878; WO 01/60970 WO 01/88197; WO 02/099084; WO98/53058; WO 98/53059; WO 98/53060; WO 02/016536 and WO 03/016496.

In addition, as disclosed in these and other references, zinc fingerdomains and/or multi-fingered zinc finger proteins may be linkedtogether using any suitable linker sequences, including for example,linkers of 5 or more amino acids in length. See, also, U.S. Pat. Nos.6,479,626; 6,903,185; and 7,153,949 for exemplary linker sequences 6 ormore amino acids in length. The proteins described herein may includeany combination of suitable linkers between the individual zinc fingersof the protein.

B. Cleavage Domains

Any suitable cleavage domain can be operatively linked to a DNA-bindingdomain to form a nuclease. For example, ZFP DNA-binding domains havebeen fused to nuclease domains to create ZFNs—a functional entity thatis able to recognize its intended nucleic acid target through itsengineered (ZFP) DNA binding domain and cause the DNA to be cut near theZFP binding site via the nuclease activity. See, e.g., Kim et al. (1996)Proc Nat'l Acad Sci USA 93(3):1156-1160. More recently, ZFNs have beenused for genome modification in a variety of organisms. See, forexample, United States Patent Publications 20030232410; 20050208489;20050026157; 20050064474; 20060188987; 20060063231; and InternationalPublication WO 07/014275.

As noted above, the cleavage domain may be heterologous to theDNA-binding domain, for example a zinc finger DNA-binding domain and acleavage domain from a nuclease or a TALEN DNA-binding domain and acleavage domain, or meganuclease DNA-binding domain and cleavage domainfrom a different nuclease. Heterologous cleavage domains can be obtainedfrom any endonuclease or exonuclease. Exemplary endonucleases from whicha cleavage domain can be derived include, but are not limited to,restriction endonucleases and homing endonucleases. See, for example,2002-2003 Catalogue, New England Biolabs, Beverly, Mass.; and Belfort etal. (1997) Nucleic Acids Res. 25:3379-3388. Additional enzymes whichcleave DNA are known (e.g., S1 Nuclease; mung bean nuclease; pancreaticDNase I; micrococcal nuclease; yeast HO endonuclease; see also Linn etal. (eds.) Nucleases, Cold Spring Harbor Laboratory Press, 1993). One ormore of these enzymes (or functional fragments thereof) can be used as asource of cleavage domains and cleavage half-domains.

Similarly, a cleavage half-domain can be derived from any nuclease orportion thereof, as set forth above, that requires dimerization forcleavage activity. In general, two fusion proteins are required forcleavage if the fusion proteins comprise cleavage half-domains.Alternatively, a single protein comprising two cleavage half-domains canbe used. The two cleavage half-domains can be derived from the sameendonuclease (or functional fragments thereof), or each cleavagehalf-domain can be derived from a different endonuclease (or functionalfragments thereof). In addition, the target sites for the two fusionproteins are preferably disposed, with respect to each other, such thatbinding of the two fusion proteins to their respective target sitesplaces the cleavage half-domains in a spatial orientation to each otherthat allows the cleavage half-domains to form a functional cleavagedomain, e.g., by dimerizing. Thus, in certain embodiments, the nearedges of the target sites are separated by 5-8 nucleotides or by 15-18nucleotides. However any integral number of nucleotides or nucleotidepairs can intervene between two target sites (e.g., from 2 to 50nucleotide pairs or more). In general, the site of cleavage lies betweenthe target sites.

Restriction endonucleases (restriction enzymes) are present in manyspecies and are capable of sequence-specific binding to DNA (at arecognition site), and cleaving DNA at or near the site of binding.Certain restriction enzymes (e.g., Type IIS) cleave DNA at sites removedfrom the recognition site and have separable binding and cleavagedomains. For example, the Type IIS enzyme Fok I catalyzesdouble-stranded cleavage of DNA, at 9 nucleotides from its recognitionsite on one strand and 13 nucleotides from its recognition site on theother. See, for example, U.S. Pat. Nos. 5,356,802; 5,436,150 and5,487,994; as well as Li et al. (1992) Proc. Natl. Acad. Sci. USA89:4275-4279; Li et al. (1993) Proc. Natl. Acad. Sci. USA 90:2764-2768;Kim et al. (1994a) Proc. Natl. Acad. Sci. USA 91:883-887; Kim et al.(1994b) J. Biol. Chem. 269:31,978-31,982. Thus, in one embodiment,fusion proteins comprise the cleavage domain (or cleavage half-domain)from at least one Type IIS restriction enzyme and one or more zincfinger binding domains, which may or may not be engineered.

An exemplary Type IIS restriction enzyme, whose cleavage domain isseparable from the binding domain, is Fok I. This particular enzyme isactive as a dimer. Bitinaite et al. (1998) Proc. Natl. Acad. Sci. USA95: 10,570-10,575. Accordingly, for the purposes of the presentdisclosure, the portion of the Fok I enzyme used in the disclosed fusionproteins is considered a cleavage half-domain Thus, for targeteddouble-stranded cleavage and/or targeted replacement of cellularsequences using zinc finger-Fok I fusions, two fusion proteins, eachcomprising a Fold cleavage half-domain, can be used to reconstitute acatalytically active cleavage domain. Alternatively, a singlepolypeptide molecule containing a DNA binding domain and two Fok Icleavage half-domains can also be used.

A cleavage domain or cleavage half-domain can be any portion of aprotein that retains cleavage activity, or that retains the ability tomultimerize (e.g., dimerize) to form a functional cleavage domain.

Exemplary Type IIS restriction enzymes are described in InternationalPublication WO 07/014275, incorporated herein in its entirety.Additional restriction enzymes also contain separable binding andcleavage domains, and these are contemplated by the present disclosure.See, for example, Roberts et al. (2003) Nucleic Acids Res. 31:418-420.

In certain embodiments, the cleavage domain comprises one or moreengineered cleavage half-domain (also referred to as dimerization domainmutants) that minimize or prevent homodimerization, as described, forexample, in U.S. Patent Publication Nos. 20050064474; 20060188987 and20080131962, the disclosures of all of which are incorporated byreference in their entireties herein. Amino acid residues at positions446, 447, 479, 483, 484, 486, 487, 490, 491, 496, 498, 499, 500, 531,534, 537, and 538 of FokI are all targets for influencing dimerizationof the FokI cleavage half-domains.

Exemplary engineered cleavage half-domains of FokI that form obligateheterodimers include a pair in which a first cleavage half-domainincludes mutations at amino acid residues at positions 490 and 538 ofFokI and a second cleavage half-domain includes mutations at amino acidresidues 486 and 499.

Thus, in one embodiment, a mutation at 490 replaces Glu (E) with Lys(K); the mutation at 538 replaces Iso (I) with Lys (K); the mutation at486 replaced Gln (Q) with Glu (E); and the mutation at position 499replaces Iso (I) with Lys (K). Specifically, the engineered cleavagehalf-domains described herein were prepared by mutating positions 490(E→K) and 538 (I→K) in one cleavage half-domain to produce an engineeredcleavage half-domain designated “E490K:1538K” and by mutating positions486 (Q→E) and 499 (I→L) in another cleavage half-domain to produce anengineered cleavage half-domain designated “Q486E:I499L”. The engineeredcleavage half-domains described herein are obligate heterodimer mutantsin which aberrant cleavage is minimized or abolished. See, e.g., U.S.Patent Publication No. 2008/0131962, the disclosure of which isincorporated by reference in its entirety for all purposes.

In certain embodiments, the engineered cleavage half-domain comprisesmutations at positions 486, 499 and 496 (numbered relative to wild-typeFold), for instance mutations that replace the wild type Gln (Q) residueat position 486 with a Glu (E) residue, the wild type Iso (I) residue atposition 499 with a Leu (L) residue and the wild-type Asn (N) residue atposition 496 with an Asp (D) or Glu (E) residue (also referred to as a“ELD” and “ELE” domains, respectively). In other embodiments, theengineered cleavage half-domain comprises mutations at positions 490,538 and 537 (numbered relative to wild-type FokI), for instancemutations that replace the wild type Glu (E) residue at position 490with a Lys (K) residue, the wild type Iso (I) residue at position 538with a Lys (K) residue, and the wild-type His (H) residue at position537 with a Lys (K) residue or a Arg (R) residue (also referred to as“KKK” and “KKR” domains, respectively). In other embodiments, theengineered cleavage half-domain comprises mutations at positions 490 and537 (numbered relative to wild-type FokI), for instance mutations thatreplace the wild type Glu (E) residue at position 490 with a Lys (K)residue and the wild-type His (H) residue at position 537 with a Lys (K)residue or a Arg (R) residue (also referred to as “KIK” and “KIR”domains, respectively). (See US Patent Publication No. 20110201055).

Engineered cleavage half-domains described herein can be prepared usingany suitable method, for example, by site-directed mutagenesis ofwild-type cleavage half-domains (Fok I) as described in U.S. PatentPublication Nos. 20050064474 and 20080131962.

Alternatively, nucleases may be assembled in vivo at the nucleic acidtarget site using so-called “split-enzyme” technology (see, e.g. U.S.Patent Publication No. 20090068164). Components of such split enzymesmay be expressed either on separate expression constructs, or can belinked in one open reading frame where the individual components areseparated, for example, by a self-cleaving 2A peptide or IRES sequence.Components may be individual zinc finger binding domains or domains of ameganuclease nucleic acid binding domain.

Nucleases can be screened for activity prior to use, for example in ayeast-based chromosomal system as described in WO 2009/042163 and U.S.Publication No. 20090068164. Nuclease expression constructs can bereadily designed using methods known in the art. See, e.g., UnitedStates Patent Publications 20030232410; 20050208489; 20050026157;20050064474; 20060188987; 20060063231; and International Publication WO07/014275. Expression of the nuclease may be under the control of aconstitutive promoter or an inducible promoter, for example thegalactokinase promoter which is activated (de-repressed) in the presenceof raffinose and/or galactose and repressed in presence of glucose.

Target Sites

As described in detail above, DNA domains can be engineered to bind toany sequence of choice in a CFTR locus or in a SFTPB locus. Anengineered DNA-binding domain can have a novel binding specificity,compared to a naturally-occurring DNA-binding domain. Engineeringmethods include, but are not limited to, rational design and varioustypes of selection. Rational design includes, for example, usingdatabases comprising triplet (or quadruplet) nucleotide sequences andindividual (e.g., zinc finger) amino acid sequences, in which eachtriplet or quadruplet nucleotide sequence is associated with one or moreamino acid sequences of DNA binding domain which bind the particulartriplet or quadruplet sequence. See, for example, co-owned U.S. Pat.Nos. 6,453,242 and 6,534,261, incorporated by reference herein in theirentireties. Rational design of TAL-effector domains can also beperformed. See, e.g., U.S. Patent Publication No. 20110301073.

Exemplary selection methods applicable to DNA-binding domains, includingphage display and two-hybrid systems, are disclosed in U.S. Pat. Nos.5,789,538; 5,925,523; 6,007,988; 6,013,453; 6,410,248; 6,140,466;6,200,759; and 6,242,568; as well as WO 98/37186; WO 98/53057; WO00/27878; WO 01/88197 and GB 2,338,237.

Selection of target sites; nucleases and methods for design andconstruction of fusion proteins (and polynucleotides encoding same) areknown to those of skill in the art and described in detail in U.S.Patent Application Publication Nos. 20050064474 and 20060188987,incorporated by reference in their entireties herein.

In addition, as disclosed in these and other references, DNA-bindingdomains (e.g., multi-fingered zinc finger proteins) may be linkedtogether using any suitable linker sequences, including for example,linkers of 5 or more amino acids. See, e.g., U.S. Pat. Nos. 6,479,626;6,903,185; and 7,153,949 for exemplary linker sequences 6 or more aminoacids in length. The proteins described herein may include anycombination of suitable linkers between the individual DNA-bindingdomains of the protein. See, also, U.S. Patent Publication No.20110287512.

Donors

As noted above, alteration of a CFTR or SFTPB gene can include insertionof an exogenous sequence (also called a “donor sequence” or “donor”),for example for correction of a mutant gene or for mutation of wild-typegene. It will be readily apparent that the donor sequence is typicallynot identical to the genomic sequence that it replaces. For example, thesequence of the donor polynucleotide can contain one or more single basechanges, insertions, deletions, inversions or rearrangements withrespect to the genomic sequence, so long as sufficient homology withchromosomal sequences is present. Alternatively, a donor sequence cancontain a non-homologous sequence flanked by two regions of homology.Additionally, donor sequences can comprise a vector molecule containingsequences that are not homologous to the region of interest in cellularchromatin. A donor molecule can contain several, discontinuous regionsof homology to cellular chromatin. For example, for targeted insertionof sequences not normally present in a region of interest, saidsequences can be present in a donor nucleic acid molecule and flanked byregions of homology to sequence in the region of interest.

The donor polynucleotide can be DNA or RNA, single-stranded ordouble-stranded and can be introduced into a cell in linear or circularform. See, e.g., U.S. Pat. No. 7,888,121 and U.S. Patent PublicationNos. 2009/0263900; 20100047805 and 20110207221, incorporated byreference herein. If introduced in linear form, the ends of the donorsequence can be protected (e.g., from exonucleolytic degradation) bymethods known to those of skill in the art. For example, one or moredideoxynucleotide residues are added to the 3′ terminus of a linearmolecule and/or self-complementary oligonucleotides are ligated to oneor both ends. See, for example, Chang et al. (1987) Proc. Natl. Acad.Sci. USA 84:4959-4963; Nehls et al. (1996) Science 272:886-889.Additional methods for protecting exogenous polynucleotides fromdegradation include, but are not limited to, addition of terminal aminogroup(s) and the use of modified internucleotide linkages such as, forexample, phosphorothioates, phosphoramidates, and O-methyl ribose ordeoxyribose residues.

A polynucleotide can be introduced into a cell as part of a vectormolecule having additional sequences such as, for example, replicationorigins, promoters and genes encoding antibiotic resistance. Moreover,donor polynucleotides can be introduced as naked nucleic acid, asnucleic acid complexed with an agent such as a liposome or poloxamer, orcan be delivered by viruses (e.g., adenovirus, AAV, herpesvirus,retrovirus, lentivirus and integrase defective lentivirus (IDLV)).

The donor is generally inserted so that its expression is driven by theendogenous promoter at the integration site, namely the promoter thatdrives expression of the CFTR gene. However, it will be apparent thatthe donor may comprise a promoter and/or enhancer, for example aconstitutive promoter or an inducible or tissue specific promoter.

Furthermore, although not required for expression, exogenous sequencesmay also be transcriptional or translational regulatory sequences, forexample, promoters, enhancers, insulators, internal ribosome entrysites, sequences encoding 2A peptides and/or polyadenylation signals.

Delivery

The nucleases, polynucleotides encoding these nucleases, donorpolynucleotides and compositions comprising the proteins and/orpolynucleotides described herein may be delivered in vivo or ex vivo byany suitable means.

Methods of delivering nucleases as described herein are described, forexample, in U.S. Pat. Nos. 6,453,242; 6,503,717; 6,534,261; 6,599,692;6,607,882; 6,689,558; 6,824,978; 6,933,113; 6,979,539; 7,013,219; and7,163,824, the disclosures of all of which are incorporated by referenceherein in their entireties.

Nucleases and/or donor constructs as described herein may also bedelivered using vectors containing sequences encoding one or more of thezinc finger or TALEN protein(s). Any vector systems may be usedincluding, but not limited to, plasmid vectors, retroviral vectors,lentiviral vectors, adenovirus vectors, poxvirus vectors; herpesvirusvectors and adeno-associated virus vectors, etc. See, also, U.S. Pat.Nos. 6,534,261; 6,607,882; 6,824,978; 6,933,113; 6,979,539; 7,013,219;and 7,163,824, incorporated by reference herein in their entireties.Furthermore, it will be apparent that any of these vectors may compriseone or more of the sequences needed for treatment. Thus, when one ormore nucleases and a donor construct are introduced into the cell, thenucleases and/or donor polynucleotide may be carried on the same vectoror on different vectors. When multiple vectors are used, each vector maycomprise a sequence encoding one or multiple nucleases and/or donorconstructs.

Conventional viral and non-viral based gene transfer methods can be usedto introduce nucleic acids encoding nucleases and donor constructs incells (e.g., mammalian cells) and target tissues. Non-viral vectordelivery systems include DNA plasmids, naked nucleic acid, and nucleicacid complexed with a delivery vehicle such as a liposome or poloxamer.Viral vector delivery systems include DNA and RNA viruses, which haveeither episomal or integrated genomes after delivery to the cell. For areview of gene therapy procedures, see Anderson Science 256:808-813(1992); Nabel&Felgner, TIBTECH 11:211-217 (1993); Mitani & Caskey,TIBTECH 11:162-166 (1993); Dillon, TIBTECH 11:167-175 (1993); Miller,Nature 357:455-460 (1992); Van Brunt, Biotechnology 6(10):1149-1154(1988); Vigne, Restorative Neurology and Neuroscience 8:35-36 (1995);Kremer &Perricaudet, British Medical Bulletin 51(1):31-44 (1995);Haddada et al., in Current Topics in Microbiology and ImmunologyDoerfler and Bohm (eds.) (1995); and Yu et al., Gene Therapy 1:13-26(1994).

Methods of non-viral delivery of nucleic acids include electroporation,lipofection, microinjection, biolistics, virosomes, liposomes,immunoliposomes, polycation or lipid:nucleic acid conjugates, naked DNA,artificial virions, and agent-enhanced uptake of DNA. Sonoporationusing, e.g., the Sonitron 2000 system (Rich-Mar) can also be used fordelivery of nucleic acids.

Additional exemplary nucleic acid delivery systems include thoseprovided by AmaxaBiosystems (Cologne, Germany), Maxcyte, Inc.(Rockville, Md.), BTX Molecular Delivery Systems (Holliston, Mass.) andCopernicus Therapeutics Inc, (see for example U.S. Pat. No. 6,008,336).Lipofection is described in e.g., U.S. Pat. Nos. 5,049,386; 4,946,787;and 4,897,355) and lipofection reagents are sold commercially (e.g.,Transfectam™ and Lipofectin™). Cationic and neutral lipids that aresuitable for efficient receptor-recognition lipofection ofpolynucleotides include those of Felgner, WO 91/17424, WO 91/16024.

The preparation of lipid:nucleic acid complexes, including targetedliposomes such as immunolipid complexes, is well known to one of skillin the art (see, e.g., Crystal, Science 270:404-410 (1995); Blaese etal., Cancer Gene Ther. 2:291-297 (1995); Behr et al., Bioconjugate Chem.5:382-389 (1994); Remy et al., Bioconjugate Chem. 5:647-654 (1994); Gaoet al., Gene Therapy 2:710-722 (1995); Ahmad et al., Cancer Res.52:4817-4820 (1992); U.S. Pat. Nos. 4,186,183, 4,217,344, 4,235,871,4,261,975, 4,485,054, 4,501,728, 4,774,085, 4,837,028, and 4,946,787).

Additional methods of delivery include the use of packaging the nucleicacids to be delivered into EnGeneIC delivery vehicles (EDVs). These EDVsare specifically delivered to target tissues using bispecific antibodieswhere one aim of the antibody has specificity for the target tissue andthe other has specificity for the EDV. The antibody brings the EDVs tothe target cell surface and then the EDV is brought into the cell byendocytosis. Once in the cell, the contents are released (see MacDiarmidet al (2009) Nature Biotechnology 27(7):643).

The use of RNA or DNA viral based systems for the delivery of nucleicacids encoding engineered ZFPs take advantage of highly evolvedprocesses for targeting a virus to specific cells in the body andtrafficking the viral payload to the nucleus. Viral vectors can beadministered directly to patients (in vivo) or they can be used to treatcells in vitro and the modified cells are administered to patients (exvivo). Conventional viral based systems for the delivery of ZFPsinclude, but are not limited to, retroviral, lentivirus, adenoviral,adeno-associated, vaccinia and herpes simplex virus vectors for genetransfer. Integration in the host genome is possible with theretrovirus, lentivirus, and adeno-associated virus gene transfermethods, often resulting in long term expression of the insertedtransgene. Additionally, high transduction efficiencies have beenobserved in many different cell types and target tissues.

The tropism of a retrovirus can be altered by incorporating foreignenvelope proteins, expanding the potential target population of targetcells. Lentiviral vectors are retroviral vectors that are able totransduce or infect non-dividing cells and typically produce high viraltiters. Selection of a retroviral gene transfer system depends on thetarget tissue. Retroviral vectors are comprised of cis-acting longterminal repeats with packaging capacity for up to 6-10 kb of foreignsequence. The minimum cis-acting LTRs are sufficient for replication andpackaging of the vectors, which are then used to integrate thetherapeutic gene into the target cell to provide permanent transgeneexpression. Widely used retroviral vectors include those based uponmurine leukemia virus (MuLV), gibbon ape leukemia virus (GaLV), SimianImmunodeficiency virus (SIV), human immunodeficiency virus (HIV), andcombinations thereof (see, e.g., Buchscher et al., J. Virol.66:2731-2739 (1992); Johann et al., J. Virol. 66:1635-1640 (1992);Sommerfelt et al., Virol. 176:58-59 (1990); Wilson et al., J. Virol.63:2374-2378 (1989); Miller et al., J. Virol. 65:2220-2224 (1991);PCT/US94/05700).

In applications in which transient expression is preferred, adenoviralbased systems can be used. Adenoviral based vectors are capable of veryhigh transduction efficiency in many cell types and do not require celldivision. With such vectors, high titer and high levels of expressionhave been obtained. This vector can be produced in large quantities in arelatively simple system. Adeno-associated virus (“AAV”) vectors arealso used to transduce cells with target nucleic acids, e.g., in the invitro production of nucleic acids and peptides, and for in vivo and exvivo gene therapy procedures (see, e.g., West et al., Virology 160:38-47(1987); U.S. Pat. No. 4,797,368; WO 93/24641; Kotin, Human Gene Therapy5:793-801 (1994); Muzyczka, J. Clin. Invest. 94:1351 (1994).Construction of recombinant AAV vectors are described in a number ofpublications, including U.S. Pat. No. 5,173,414; Tratschin et al., Mol.Cell. Biol. 5:3251-3260 (1985); Tratschin, et al., Mol. Cell. Biol.4:2072-2081 (1984); Hermonat & Muzyczka, PNAS 81:6466-6470 (1984); andSamulski et al., J. Virol. 63:03822-3828 (1989).

At least six viral vector approaches are currently available for genetransfer in clinical trials, which utilize approaches that involvecomplementation of defective vectors by genes inserted into helper celllines to generate the transducing agent.

pLASN and MFG-S are examples of retroviral vectors that have been usedin clinical trials (Dunbar et al., Blood 85:3048-305 (1995); Kohn etal., Nat. Med. 1:1017-102 (1995); Malech et al., PNAS 94:22 12133-12138(1997)). PA317/pLASN was the first therapeutic vector used in a genetherapy trial. (Blaese et al., Science 270:475-480 (1995)). Transductionefficiencies of 50% or greater have been observed for MFG-S packagedvectors. (Ellem et al., Immunol Immunother. 44(1):10-20 (1997); Dranoffet al., Hum. Gene Ther. 1:111-2 (1997).

Recombinant adeno-associated virus vectors (rAAV) are a promisingalternative gene delivery systems based on the defective andnonpathogenic parvovirus adeno-associated type 2 virus. All vectors arederived from a plasmid that retains only the AAV 145 bp invertedterminal repeats flanking the transgene expression cassette. Efficientgene transfer and stable transgene delivery due to integration into thegenomes of the transduced cell are key features for this vector system.(Wagner et al., Lancet 351:9117 1702-3 (1998), Kearns et al., Gene Ther.9:748-55 (1996)). Other AAV serotypes, including AAV1, AAV3, AAV4, AAV5,AAV6, AAV8, AAV9, and AAVrh10 can also be used in accordance with thepresent invention. Additionally, pseudotyped AAV vectors may be usedwherein the AAV vector ITRs and the AAV capsid proteins are fromdifferent AAV serotypes, or chimeric AAV particles where the capsidproteins are made from more than one AAV serotype.

Replication-deficient recombinant adenoviral vectors (Ad) can beproduced at high titer and readily infect a number of different celltypes. Most adenovirus vectors are engineered such that a transgenereplaces the Ad E1a, E1b, and/or E3 genes; subsequently the replicationdefective vector is propagated in human 293 cells that supply deletedgene function in trans. Ad vectors can transduce multiple types oftissues in vivo, including non-dividing, differentiated cells such asthose found in liver, kidney and muscle. Conventional Ad vectors have alarge carrying capacity. An example of the use of an Ad vector in aclinical trial involved polynucleotide therapy for anti-tumorimmunization with intramuscular injection (Sterman et al., Hum. GeneTher. 7:1083-9 (1998)). Additional examples of the use of adenovirusvectors for gene transfer in clinical trials include Rosenecker et al.,Infection 24:1 5-10 (1996); Sterman et al., Hum. Gene Ther. 9:71083-1089 (1998); Welsh et al., Hum. Gene Ther. 2:205-18 (1995); Alvarezet al., Hum. Gene Ther. 5:597-613 (1997); Topf et al., Gene Ther.5:507-513 (1998); Sterman et al., Hum. Gene Ther. 7:1083-1089 (1998).

Packaging cells are used to form virus particles that are capable ofinfecting a host cell. Such cells include 293 cells, which packageadenovirus, and ψ2 cells or PA317 cells, which package retrovirus. Viralvectors used in gene therapy are usually generated by a producer cellline that packages a nucleic acid vector into a viral particle. Thevectors typically contain the minimal viral sequences required forpackaging and subsequent integration into a host (if applicable), otherviral sequences being replaced by an expression cassette encoding theprotein to be expressed. The missing viral functions are supplied intrans by the packaging cell line. For example, AAV vectors used in genetherapy typically only possess inverted terminal repeat (ITR) sequencesfrom the AAV genome which are required for packaging and integrationinto the host genome. Viral DNA is packaged in a cell line, whichcontains a helper plasmid encoding the other AAV genes, namely rep andcap, but lacking ITR sequences. The cell line is also infected withadenovirus as a helper. The helper virus promotes replication of the AAVvector and expression of AAV genes from the helper plasmid. The helperplasmid is not packaged in significant amounts due to a lack of ITRsequences. Contamination with adenovirus can be reduced by, e.g., heattreatment to which adenovirus is more sensitive than AAV.

In many gene therapy applications, it is desirable that the gene therapyvector be delivered with a high degree of specificity to a particulartissue type. Accordingly, a viral vector can be modified to havespecificity for a given cell type by expressing a ligand as a fusionprotein with a viral coat protein on the outer surface of the virus. Theligand is chosen to have affinity for a receptor known to be present onthe cell type of interest. For example, Han et al., Proc. Natl. Acad.Sci. USA 92:9747-9751 (1995), reported that Moloney murine leukemiavirus can be modified to express human heregulin fused to gp70, and therecombinant virus infects certain human breast cancer cells expressinghuman epidermal growth factor receptor. This principle can be extendedto other virus-target cell pairs, in which the target cell expresses areceptor and the virus expresses a fusion protein comprising a ligandfor the cell-surface receptor. For example, filamentous phage can beengineered to display antibody fragments (e.g., FAB or Fv) havingspecific binding affinity for virtually any chosen cellular receptor.Although the above description applies primarily to viral vectors, thesame principles can be applied to nonviral vectors. Such vectors can beengineered to contain specific uptake sequences which favor uptake byspecific target cells.

Gene therapy vectors can be delivered in vivo by administration to anindividual patient, typically by systemic administration (e.g.,intravenous, intraperitoneal, intramuscular, subdeinial, or intracranialinfusion) or topical application, as described below. Alternatively,vectors can be delivered to cells ex vivo, such as cells explanted froman individual patient (e.g., lymphocytes, bone marrow aspirates, tissuebiopsy) or universal donor hematopoietic stem cells, followed byreimplantation of the cells into a patient, usually after selection forcells which have incorporated the vector.

Vectors (e.g., retroviruses, adenoviruses, liposomes, etc.) containingnucleases and/or donor constructs can also be administered directly toan organism for transduction of cells in vivo. Alternatively, naked DNAcan be administered. Administration is by any of the routes normallyused for introducing a molecule into ultimate contact with blood ortissue cells including, but not limited to, injection, infusion, topicalapplication and electroporation. Suitable methods of administering suchnucleic acids are available and well known to those of skill in the art,and, although more than one route can be used to administer a particularcomposition, a particular route can often provide a more immediate andmore effective reaction than another route. In particular, for deliveryto pulmonary tissues, introduction of vectors may be done using thebronchial artery. Bronchial delivery may occur in conjunction with stopflow techniques, or with the use of endothelial barrier disrupters (e.g.VEGF or histamine) to increase uptake.

Vectors suitable for introduction of polynucleotides described hereininclude non-integrating lentivirus vectors (IDLY). See, for example, Oryet al. (1996) Proc. Natl. Acad. Sci. USA 93:11382-11388; Dull et al.(1998) J. Virol. 72:8463-8471; Zuffery et al. (1998) J. Virol.72:9873-9880; Follenzi et al. (2000) Nature Genetics 25:217-222; U.S.Patent Publication No 2009/0054985.

Pharmaceutically acceptable carriers are determined in part by theparticular composition being administered, as well as by the particularmethod used to administer the composition. Accordingly, there is a widevariety of suitable formulations of pharmaceutical compositionsavailable, as described below (see, e.g., Remington's PharmaceuticalSciences, 17th ed., 1989).

It will be apparent that the nuclease-encoding sequences and donorconstructs can be delivered using the same or different systems. Forexample, a donor polynucleotide can be carried by a plasmid, while theone or more nucleases can be carried by a AAV vector. Furthermore, thedifferent vectors can be administered by the same or different routes(intramuscular injection, tail vein injection, other intravenousinjection, intraperitoneal administration and/or intramuscularinjection. The vectors can be delivered simultaneously or in anysequential order.

Formulations for both ex vivo and in vivo administrations includesuspensions in liquid or emulsified liquids. The active ingredientsoften are mixed with excipients which are pharmaceutically acceptableand compatible with the active ingredient. Suitable excipients include,for example, water, saline, dextrose, glycerol, ethanol or the like, andcombinations thereof. In addition, the composition may contain minoramounts of auxiliary substances, such as, wetting or emulsifying agents,pH buffering agents, stabilizing agents or other reagents that enhancethe effectiveness of the pharmaceutical composition.

Applications

The instant invention describes methods and compositions that can beused to introduce or repair mutations in lung disorders such as CFdisease and/or SB-P deficiency. In particular, specific mutations at theCFTR gene that have been shown to be pathogenic in the development of CFinclude ΔF508 and ΔI507. Mutations in SFTPB are the most commonmutations leading to SB-P deficiency and include: 121ins2(121C>GAA).Thus, the methods and compositions of the instant invention are usefulfor repairing (correcting) mutations in CFTR and/or SB-P either byrepair of patient derived stem cells or by in vivo administration ofnucleases and donor molecule. Also useful described herein are methodsfor developing cell and transgenic animal models to study theintracellular pathology associated with CFTR and SFTPB mutations and forstudying the consequences of these mutations within the whole organism.As such, tools designed to knock out, knock in and/or correct specificCFTR or SFTPB mutations (for example the ΔF508 mutation in CFTR) can beused to create cell and animal models useful in furthering anunderstanding of the underlying biology and in the development ofspecific drug therapies. Further, specific nucleases targeted to aspecific CFTR or SFTPB mutation can be employed to knock out or correctthe mutation. Nucleases can also be used to cause the insertion of aCFTR or SFTPB mutation-specific tag in order to develop cell lines forthe investigation of CFTR or SFTPB mutation specific therapeutics.

Additionally, cells, cell lines and transgenic animals as describedherein are useful for drug development. Such cells and animals mayreveal phenotypes associated with a particular mutation (e.g. CFTRΔF508) or with its correction, and may be used to screen drugs that willinteract either specifically with the mutation(s) in question, or thatare useful for treatment of the disease in an afflicted animal.Therapeutically, iPSCs can be derived ex vivo from a patient afflictedwith a known genetic mutation associated with CF or SB-P deficiency, andthe mutation can be corrected using ZFN- or TALEN-mediated genecorrection. Similarly, lung, skin or other stem cells may be isolatedfrom a patient and then corrected at the CFTR or SFTPB locus using themethods and compositions of the invention. The corrected stem cells canthen be used to treat the patient. In addition, cell lines can be madefrom patient samples containing the CFTR or SFTPB mutations of interest.These cell lines can provide tools to investigate the effects ofspecific mutations in patient-specific iPS cell lines. For example,parallel cell lines can be generated in which one line is corrected atthe mutation of interest while its parallel line is not. This createscell lines that are only different by the disease-causing mutation. Theresulting isogenic panel of iPSCs that carry different allelic forms ofCFTR or SFTPB at the endogenous locus provides a genetic tool for repairof disease-specific mutations, drug screening and discovery, and diseasemechanism research.

The availability of patient-specific iPS cell lines with both repairedand induced mutations and their isogenic controls are also useful in awide-variety of medical applications, including but not limited to, thestudy of mechanisms by which these mutations cause disease andtranslating “laboratory cures” to treatments for patients who actuallymanifest disease induced by these mutations. In addition, the lines maybe useful in screening potential therapeutic compounds to identify thosecompounds that exhibit highly specific behavior.

Cellular transplantation of lung stem/progenitor cells represents apotential therapeutic approach for a variety of inherited monogenic lungdiseases such as CF or SB-P deficiency. Corrected CF or SB-P iPS cellspresent a potential source of patient-specific cells capable, in vitro,of differentiation into various lung stem/progenitor cells (see, e.g.,Chen et al. (2009) Proc Am Thorac Soc 6:602-606; Kajstura et al. (2011)N Engl J Med 364:1795-1806; either for transplantation of autologouslung cells or for seeding de-vitalized lung scaffolds ex vivo togenerate autologous lungs (see, e.g., Ott et al. (2010) Nat Med16:927-933). In addition, there are reports (see Kajstura et al, ibid)that human lung stem cells have been identified which are capable offorming bronchioles, aveoli, and pulmonary vessels when given to micewith damaged lungs in vivo. Thus there is a potential that lung or othertypes of stem cells may be able to be isolated from patients, modifiedby ZFNs or TALENs ex vivo, and then reintroduced to the patient, thustreating the disease. Thus, the methods and compositions describedherein can be used to generate cells (and their progeny) for use intransplantation that are corrected (both genotypically andphenotypically) for the CF or SB-P deficiency disease-causing mutation.These transplanted cells would not elicit an immune response in therecipient. Using skin or blood cells from affected patients, autologousinduced pluripotent stem (iPS) cells are derived. Utilizingsite-specific homology-directed repair, the disease-causing mutationwould then be corrected in the endogenous, chromosomal DNA sequence.Finally, a directed differentiation approach would be employed to obtainhighly purified populations of the relevant lung stem/progenitor cellsfrom the corrected iPS cells for purposes of transplantation.

The following Examples relate to exemplary embodiments of the presentdisclosure in which the nuclease comprises a zinc finger nuclease (ZFN)or a TALEN. It will be appreciated that this is for purposes ofexemplification only and that other nucleases can be used, for instancehoming endonucleases (meganucleases) with engineered DNA-binding domainsand/or fusions of naturally occurring of engineered homing endonucleases(meganucleases)

EXAMPLES Example 1 Materials and Methods A. Cystic Fibrosis PrimaryFibroblasts

CF primary fibroblast line GM04320 was obtained (Coriell Repository,Camden, N.J.) from a patient (17 year old male) reported homozygous forthe ΔF508 mutation. Clinical symptoms for this patient were reported asadvanced pulmonary disease and pancreatic insufficiency; in addition,defective cAMP stimulated chloride channel activity was demonstrated infibroblasts from this patient. See, also, Lin & Gruenstein (1987) J BiolChem 262:15345-15347.

Sequencing of the CFTR alleles in genomic DNA isolated from the GM04320fibroblasts demonstrated that the patient was, in fact, a compoundheterozygote with one allele being ΔF508 and the other ΔI507.ΔF508/ΔI507 compound heterozygosity has previously been reported in CFpatients. See, e.g., Kerem et al. (1990) Proc Natl Acad Sci USA87:8447-8451.

B. CF iPS Cell Generation and Characterization

The pMXs retroviral vectors encoding human reprogramming factors (OCT4[17964], SOX2 [17965], KLF4 [17967], C-MYC [17966], NANOG [18115]) asdescribed in Lowry et al. (2008) Proc Natl Acad Sci USA 105:2883-2888were introduced into the CF primary fibroblasts. Non-integrating methodsor integration-free methods (e.g. RNA, episomal vectors, excisablereprogramming transgenes, and/or small molecules) can also be employedfor introducing of reprogramming factors. See, e.g., Somers et al.(2010) Stem Cells 28:1728-1740; Warren et al. (2010) Cell Stem Cell7:618-630; Yu et al. (2009) Science 324:797-801; Li et al. (2009) CellStem Cell 4:16-19. VSV-G enveloped viral stocks were prepared bytransfection of Plat-GP cells (Cell Biolabs) with vector DNA and VSV-Gexpression plasmids (pCMV-VSV-G [8454]) and concentrated 100 fold byultracentrifugation. Parallel production of pMXs-GFP vector stocks wasperformed; titration of the pMXs-GFP virus was performed by infection ofprimary human fibroblasts and subsequent FACS analysis forGFP-expressing cells.

CF fibroblasts, plated at 10⁵ cells per well of a 6-well plate on day 0,were transduced on days 1 and 2 by spinfection (200×g for 30 minutes) ata multiplicity of infection of 21.5, in the presence of 10 micrograms/mlprotamine sulfate. On day 4, fibroblasts were transferred ontoirradiated mouse embryo fibroblasts (MEFs; CF-1 mouse strain, CharlesRiver), and one day later media was switched to human embryonic stem(ES) cell media (per National Stem Cell Bank protocol SOP-CC-001Cavailable on the internet) containing 40 ng/ml basic Fibroblast growthfactor (bFGF) and re-fed daily. Starting on day 12, cells were re-feddaily with human ES cell media pre-conditioned on irradiated MEFs.Beginning at 16 days post transduction, iPS-like colonies were firstidentified based on morphological criteria. Live-cell staining witheither Alexa 488-conjugated anti-Tra-1-60 monoclonal antibody (Stemgent)or anti-Tra-1-81 monoclonal antibody (Millipore) followed by Alexa 488goat anti-mouse IgM (Invitrogen) was then used to identify reprogrammedcolonies for subsequent expansion and characterization. Of 32 coloniesoriginally picked (all of which stained positive for Tra-1-60 and/orTra-1-81), 9 colonies were subsequently expanded and cryopreserved andtwo iPS clones (clones 17 and 28) were selected for more extensivecharacterization.

CF iPS cells were stained for expression of Oct4 and SSEA-4 per NSCBprotocol SOP-CH-102C, and analyzed either by fluorescence microscopy orby fluorescence activated cell analysis (LSR-II, Becton Dickinson).Co-staining with anti-CD29 (FITC-conjugated; Source) was used to excludecontaminating MEFs. Non-specific alkaline phosphatase activity was alsoassessed (Vector Lab). Karyotyping of CF iPS clones 17 (passages 5 and17) and 28 (passages 5 and 13) was performed at Texas Children'sHospital Clinical and Research Cytogenetic Laboratory. Genomic DNA wasisolated from CF iPS clones 17 and 28; sequences containing exon 10 wereamplified by PCR and sequenced.

C. Teratoma Assay

CF iPS cells (clone 17) were injected intra-muscularly into the reardorsal leg of four week old Fox Chase SCID beige mice (Charles River)and monitored weekly for the appearance of tumor growth. At seven weekspost injection, tumors were removed, paraffin embedded, prepared forhistological examination by hematoxylin and eosin, and analyzed by theCenter for Comparative Medicine at Baylor College of Medicine.

D. ZFN-Mediated Targeting

Zinc finger nucleases targeted to CFTR were engineered essentially asdescribed in U.S. Pat. No. 6,534,261. Table 1 shows the recognitionhelices DNA binding domain of exemplary CFTR-targeted ZFPs. The designedDNA-binding domains contain four to six zinc fingers, recognizingspecified target sequences (see Table 2). Nucleotides in the target sitethat are contacted by the ZFP recognition helices are indicated inuppercase letters; non-contacted nucleotides indicated in lowercase.

TABLE 1 CFTR Zinc Finger Nucleases Design SBS # F1 F2 F3 F4 F5 F6 12897WPSCLYA NGVLLKR QSGNLAR RSDNLSE NPRNRFT N/A (SEQ ID (SEQ ID (SEQ ID(SEQ ID (SEQ ID NO: 8) NO: 9) NO: 10) NO: 11) NO: 12) 9940 RSDVLSEQSGNLAR QSGHLSR RSDVLSE WSASLSK N/A (SEQ ID (SEQ ID (SEQ ID (SEQ ID(SEQ ID NO: 13) NO: 10) NO: 14) NO: 15) NO: 16) 32365 QNATRIN QSGNLARRSDNLST QSADRKK N/A N/A (SEQ ID (SEQ ID (SEQ ID (SEQ ID NO: 17) NO: 10)NO: 18) NO: 19) 32366 TNQNRIT RNQTRIT QSGNLAR QSNTRIM TSGNLTR QSNALHQ(SEQ ID (SEQ ID (SEQ ID (SEQ ID (SEQ ID (SEQ ID NO: 20) NO: 21) NO: 10)NO: 22) NO: 23) NO: 24) 32375 TSSDRKK QSSDLSR DRSNLTR TSGNLTR WRLSLQVN/A (SEQ ID (SEQ ID (SEQ ID (SEQ ID (SEQ ID NO: 25) NO: 26) NO: 27)NO: 23) NO: 28) 32376 QSGNLAR QGANLIK RSDHLSA ESRYLMV RSDNLST  DRSNRKT(SEQ ID (SEQ ID (SEQ ID (SEQ ID (SEQ ID (SEQ ID NO: 10) NO: 29) NO: 30)NO: 31) NO: 18) NO: 32) 32401 TSGNLTR QSNALHQ QSGNLAR TSGNLTR WWTSRALN/A (SEQ ID (SEQ ID (SEQ ID (SEQ ID (SEQ ID NO: 23) NO: 24) NO: 10)NO: 23) NO: 33) 32398 HSNARKT TSGNLTR TLQNRMS DQSTLRN N/A N/A (SEQ ID(SEQ ID (SEQ ID (SEQ ID NO: 34) NO: 23) NO: 35) NO: 36)

TABLE 2 CFTR Target sites SBS # Target Site 12897atTAGAAGtGAAGTCTGGaaataaaacc(SEQ ID NO: 37) 9940agtgATTATGGGAGAACTGgatgttcacagtcagtccacacgtc(SEQ ID NO: 38) 32365caTCATAGGAAACAccaaagatgatatt(SEQ ID NO: 39) 32366atATAGATACAGAAgCGTCATcaaagca (SEQ ID NO: 40) 32375gcTTTGATGACGCTTCTgtatctatatt (SEQ ID NO: 41) 32376ccAACTAGAAGAGGTAAGAAactatgtg(SEQ ID NO: 42) 32401ccTATGATGAAtATAGATacagaagcgt(SEQ ID NO: 43) 32398acACCAATGATATTttctttaatggtgc(SEQ ID NO: 44)

ZFN pair 12897/9940 was constructed by fusing the desired DNA bindingmotifs to the cleavage domain of the Fok I endonuclease. ZFNs weredelivered to cells either in the form of a DNA expression plasmid or invitro generated RNA (Epicentre and Ambion). A 1.6 kb donor containingwild-type exon 10 sequences (approximately 860 bp and 290 bp of homologysequences upstream and downstream of exon 10, respectively) wasoriginally constructed by PCR amplification of genomic DNA sequencesfrom BAC clone RP11-1152A23. Two silent single base pair substitutionswere introduced into the right ZFN binding site with the goal ofinterfering the ability of the introduced ZFNs to cleave the donoreither prior or subsequent to homology-directed repair; an additionalsilent single base pair substitution was introduced into the wild-typeexon 10 donor sequences in order to create a novel Cla I restrictionenzyme site. Three additional single base pair changes were introducedinto intron 10 donor sequences 125 bp downstream of exon 10 to create aunique Avr II restriction enzyme site. All changes in the wild-typedonor were introduced via Quikchange® Lightning Site-DirectedMutagenesis (Agilent). PCR amplification of pgk-puroTK-bpA sequencesfrom plasmid pPthC-Oct3/4 (see, e.g., Masui et al. (2007) Nat Cell Biol9:625-635) with primers including loxP recognition sequences and Avr IIsites generated material for cloning into the introduced Avr II site inthe CFTR donor.

ZFNs, either in the form of DNA expression plasmids (1 or 2 micrograms)or in vitro transcribed RNA (1.5 or 3 micrograms), were deliveredtogether with donor DNA (4 or 8 micrograms) to CF iPS cells (2 millioncells, cells obtained via Accutase™ treatment; clone 17) vianucleofection (Amaxa program A23) and cells were plated in the presenceof 10 microM Rock-inhibitor (Alexis Biochemicals, Y27632) ontopuromycin-resistant irradiated MEFs. Puromycin selection (0.5 microgramsper ml) was initiated four days post transfection, andpuromycin-resistant colonies were picked starting 2-5 days later andexpanded, in the presence of puromycin, to establish clonal cell lines.

E. Molecular Analysis of Targeted iPS Clones

Genomic DNA was isolated from puromycin-resistant clones beginning atpassage 2 by QIAprep® Spin Miniprep Kit (Qiagen) or ArchivePure™ DNACell Tissue Kit (5 Prime). PCR amplification utilizing various primerswas performed according to manufacturer protocols. Sequencing wasperformed on an ABI 3730XL sequencer.

F. Southern Blotting

In order to generate a radio-labeled DNA for probing Southern blottedgenomic DNAs, the donor plasmid was digested with NdeI+SpeI, separatedon 0.8% agarose gel, and then the 2.3 kb fragment was cut out andgel-purified (Qiagen). The 2.3 kb fragment was labeled with [α-³²P]dCTPusing Prime-It® II Random Primer Labeling kit (Agilent Technologies)following manufacturer's instruction. 25 micrograms of genomic DNAs(gDNAs) were digested with SpeI overnight and purified byphenol/chloroform extraction. The gDNAs were then resolved on 1% agarosegel and transferred to a Nytran® Super Charge membrane (Schleicher andSchuell) and hybridized with ³²P-labeled probe. The membrane was exposedand image scanned using a phosphorimager system (Molecular Dynamics).

G. Cre-Mediated Excision of Selectable Marker

Cre-expression plasmids (pBS513 EF1alpha-cre and pCAG-Cre (see, e.g., Leet al. (1999) Anal Biochem 270:334-336; Matsuda & Cepko (2007)Proc NatlAcad Sci USA 104:1027-1032) were delivered to Accutase™-treated cellsvia Amaxa™ nucleofection and plated onto irradiated MEFs. Individualcolonies were picked and expanded, and then plated in replicate toidentify those clones that had become sensitive to puromycin.Alternatively, some clones were first identified based on theirresistance to FIAU (1 microM, Moravek Biochemicals), expanded, and thenplated in replicate to identify puromycin sensitive clones.

H. Analysis of mRNA

RNA isolation from iPS and iPS-derived cells with the RNeasy® kit(Qiagen), cDNA synthesis was performed with Improm-II™ ReverseTranscriptase oligodT kit (Promega), and RT-PCR was performed with GotaqHot Start® polymerase (Promega).

I. In Vitro Differentiation

Short-term differentiation of iPS cells to definitive endoderm wasconducted essentially as described in D'Amour et al. (2005) NatBiotechnol 23:1534-1541. In brief, iPS cells, plated on MEFs, wereexposed to Activin A (100 micrograms/ml) in the presence of lowconcentrations of fetal bovine serum (0& on day 0, 0.2% day 1, 2% days2-5). Cultures were harvested for RNA on the indicated days and analyzedby RT-PCR for gene expression. For longer-term differentiation, weadapted the air liquid interface protocol reported by Van Haute et al.(2009) Respir Res 10:105 to generate lung epithelial tissue from humanES cells. In brief, iPS cells recovered by collagenase digestion, wereplated as clumps of cells onto 8.0 micron culture plate inserts(P18P01250, Millipore) in wells of a 12-well plate previously platedwith irradiated MEFs. For the first 8 days (days 0-4: human ES media;days 5-8: Differentiation Media [DM]: human ES media withoutbeta-mercaptoethanol and basic Fibroblast growth factor) media wasmaintained at a level sufficient to completely cover the membrane-platedcells; from days 8 to 28, the media (DM) volume was reduced to providethe desired air liquid interface. Cultures were harvested for RNA on theindicated days and analyzed by RT-PCR for gene expression.

J. Testing of Nucleases Targeted to CFTR Mutations

ZFN pairs shown in Table 1 that were designed to be close the Δ508mutation site include ZFN pair 12897/9940, that binds approximately 115nt away from the site of Δ508, and pairs 32365/32366 and 32375/32376which target sites are 18 and 48 nucleotides away, respectively. See,FIG. 6. In addition, pair 32401/32398 were designed to specificallytarget the Δ508 allele and not the wild type allele (see FIG. 6).

These indicated pairs were tested for nuclease activity in K562 cellsand the 32365/32366 pair was found to cause a 9% measure of indelformation, and the 32375/32376 caused 12% indels. The 32401/32398 pairwere tested in using the DLSSA reporter system in Neuro2A cells (seeco-owned US Publication 20110301073). In this assay, the 32401/32398pair gave a ratio of 1.45 firefly luminescence/renilla luminescence(compared to 0.16 for the pVAX vector control), demonstrating that allpairs were active.

Example 2 Derivation and Characterization of CF iPS Cells

CF primary fibroblasts (GM04320; Coriell Repository) were obtained froma patient reported homozygous for the ΔF508 CFTR mutation. Directsequencing of exon 10 revealed these cells are actually compoundheterozygous at the CFTR locus, with one allele ΔF508 and the otherallele ΔI507.

Utilizing VSV-G pseudotyped pMXs retroviral vectors encodingreprogramming factors (OCT4, SOX2, KLF4, C-MYC, NANOG), we transducedthe CF skin fibroblasts, transferred them onto mouse embryo fibroblasts(MEFs), and selected for reprogrammed cells in human ES cell media asdescribed in Example 1. Beginning at 16 days post transduction, iPS-likecolonies were first identified based on morphological criteria.Live-cell staining with anti-Tra-1-60 and/or anti-Tra-1-81 antibodieswas then used to identify successfully reprogrammed colonies forsubsequent expansion and characterization. We identified ninereprogrammed colonies, which we expanded further and cryopreserved.

Two clones (nos. 17 and 28) were selected for further study. Theseclones exhibited morphology and growth properties consistent with hEScells; we verified ΔI507/ΔF508 compound heterozygosity in the derivediPS cells. We also confirmed by immuno-staining that these two clonesexpress cellular antigens characteristic of undifferentiated hES cells.By FACS analysis, we demonstrated co-expression of Oct4 and SSEA4antigens by 90% of iPS cells for at least 33 passages. The pluripotencyof the CF iPS cells was demonstrated via teratoma assay; cell typescharacteristic of mesoderm, ectoderm, and endoderm were present inrecipient mice. We also confirmed these CF iPS cells have a normalkaryotype.

Example 3 Correction of CFTR Mutation Via ZFN-Mediated HDR

Our overall strategy for correction of CFTR exon 10 mutations isoutlined in FIG. 1 and included delivering CFTR-specific ZFNs togetherwith an appropriate CFTR donor DNA; the loxP-flanked puroTK selectablecassette permits puromycin-mediated selection of initial clones as wellas subsequent FIAU-mediated selection of Cre-excised clones. We designedZFNs targeting CFTR exon 10 to facilitate the correction of either ΔI507or ΔF508 by HDR (see, FIG. 1). The CFTR exon 10-specific ZFNs (CFTRZFNs) recognize DNA sequences close to the start of exon 10,approximately 110 bp upstream of either the ΔI507 or ΔF508 three bpdeletions.

The CFTR DNA donor repair template included a total of approximately 1.6kb of flanking homologous sequence; the donor-encoded exon 10 sequencewas modified to include three silent by substitutions: two in the ZFN-Rtarget sequence to prevent ZFN re-cleavage of the corrected CFTR locus;and a silent mutation 22 bp downstream of the restored three bpwild-type sequence to create a novel Cla I restriction enzyme site forthe rapid identification of gene edited cells by PCR. In addition, theloxP-flanked pgk-puroTK selectable cassette was inserted, in ananti-sense orientation, into intron 10 of the donor, 125 bp downstreamof the end of exon 10. Thus, the desired CFTR gene editing event wouldinvolve both the correction of the three base pair deletion in exon 10at either the ΔI507 or ΔF508 alleles as well as targeted insertion ofthe selection cassette in intron 10.

The CFTR ZFNs, either in the form of DNA expression plasmids or in vitrotranscribed RNA, were co-delivered with a plasmid encoding the CFTRdonor to CF iPS cells as described in Example 1. Puromycin-resistantcolonies were initially screened via PCR and then sequenced to confirmCFTR exon 10 was corrected via HDR (FIG. 2). The initial PCR screenassayed for targeted insertion of the puromycin selectable marker intointron 10 of the endogenous CFTR locus with one primer annealing to asequence upstream in intron 9 (not present in the donor) and one primerannealing within the loxP-flanked selectable marker (FIG. 2).

This initial screen identified potential targeted insertion events atone of the CFTR alleles in 33% (21 out of 64 clones analyzed) ofpuromycin resistant colonies. This amplicon was completely digested withCla I, consistent with incorporation of the wild-type exon 10 donorsequence. The amplicon was further analyzed by direct sequencing toverify the edited allele encoded the donor-derived corrective sequenceinstead of the ΔI507 or ΔF508 mutant genotypes. In addition, the silentbase pair substitutions introduced into the donor at the recognitionsite for the right-hand ZFN were also present in the corrected allele.

Utilizing primers that anneal to sequences outside the region ofhomology encoded by the donor, we selectively amplified by PCR onlyunmodified CFTR alleles from each of the twenty-one clones. Sequencingof the unmodified allele yielded pure sequence containing either theΔI507 mutant allele (present in 3 of 21 clones) or ΔF508 mutant allele(in 18 of 21 clones) in all clones; this result is consistent withtargeted insertion of the selectable marker occurring at only one CFTRallele per clone.

The results of two gene editing experiments performed independently areshown in Table 3, where “TI” refers shows targeted integration.

TABLE 3 ZFN-mediated genomic editing of CFTR # clones # clones modifiedmodified (Exp't 1) (Exp't 2) 64 total puroR colonies analyzed 15  49  21(33%) met 1/1′ criterion [3 TI in 10 (2 TI in 11 (1 TI in ΔF508, 18 TIin ΔI507] ΔF508) ΔF508) 7 (11%) satisfied 1/1′ and 2/2′ criteria 2 5[all TI in DI507] 4 satisfied 1/1′, 2/2′, and cDNA criteria 2 2 [all TIin DI507]

We further analyzed the 21 clones satisfying the 1/1′ criterion by PCRamplification with one primer annealing to the loxP-flanked selectablemarker and the other primer to a site downstream in intron 10 outside ofthe donor sequences.

As shown in FIG. 2, this analysis confirmed targeted insertion of theselectable marker in seven of the 21 previously identified clones.

As shown in Table 3, HDR-driven genome editing occurred more frequentlyat the ΔI507 CFTR allele than the ΔF508 allele. Accordingly, we alsofully sequenced the 1.6 kb endogenous CFTR sequences, corresponding tothe donor, of each mutant allele (ΔI507 or ΔF508) to examine whetherthere was any increased similarity of donor sequence to either mutantallele (perhaps favoring HDR in one allele vs. the other. sequencinganalysis). This sequence analysis revealed a single base pairsubstitution (A>G) in intron 9, 76 bp upstream of the ZFN cleavage site(A>G substitution occurs at position −61 in intron 9 with respect tostart of exon 10:i.e. −61A>G, present in the ΔF508 mutant allele, butabsent in both the ΔI507 mutant allele and the donor. As this singlebase pair difference occurring selectively in the ΔF508 allele of the CFiPS cells may have caused a significant decrease in the efficiency ofhomology pairing and strand invasion of the ΔF508 allele and donortemplate. Accordingly, introducing this A>G mutation into donorsequences is expected to favor targeted correction of the ΔF508 allele.

Example 4 Expression of the Corrected CFTR Gene in Gene Edited iPS Cellsand iPS-Derived Cells

Expression of CFTR in ZFN-edited cells was also determined by RT-PCR andsequencing analysis. See, Example 1.

As shown in FIG. 3A, and in agreement with quantitative expressionanalysis of other human ES/iPS cell lines (see, e.g. Bock et al. (2011)Cell 144:439-452, we detected CFTR expression in the original,uncorrected ΔI507/ΔF508 Clone 17 iPS cells by RT-PCR. Sequencingdemonstrated nearly equal levels of CFTR mRNA expression from both theΔI507 and ΔF508 alleles (FIG. 3B). As a control, analysis of the A549lung epithelial cell line confirmed wild-type CFTR expression (FIGS. 3Aand 3B). As shown in FIG. 3A, RT-PCR analysis for four of the seventargeted iPS lines (Clones 17-1, 17-9, 17-14, 17-16) yielded a singleband of similar size to that seen for the ΔI507/ΔF508 Clone 17 iPS andA549 cell lines; three of the seven clones (Clones 17-13, 17-17, 17-20)also exhibited a second RT-PCR band of greater size and were no longerconsidered for analysis.

Sequencing confirmed the expected cDNA organization (exon 9-exon 10 exon11) and demonstrated CFTR expression arising from both the non-targetedmutant allele (ΔF508) as well as the corrected allele. See, also, FIG.3B. In particular, we consistently observed expression of the correctedallele was approximately 25-35% of the unmodified mutant allele. CFTRgenomic DNA exon 10 sequences at both targeted (in all cases ΔI507) andunmodified alleles (ΔF508) for each of these four clones were determinedand Southern blot analysis was performed utilizing a pgk-puroTK probe toconfirm the correct genomic organization in the corrected CFTR alleleand to identify whether any of the successfully edited iPS clones alsoexhibited off-target integration of donor sequences.

This analysis revealed that these four clones (17-1, 17-9, 17-14, 17-16)were successfully edited without any additional integration of donorsequences; two of the clones previously eliminated (17-17 and 17-20)based on RT-PCR (see above) revealed additional pgk-puroTK integrations.Karyotypic analysis was performed on two of the correctly edited clones(17-9, 17-16) and revealed that both clonal lines exhibited a normalkaryotype.

Sequencing also demonstrated all four cell lines had equivalent levelsof putative novel single nucleotide variations (SNV) compared to thereference genome with 1,155 SNV in mutant CF fibroblast, 1,127 SNV inmutant iPS cells (Clone 17), 1,180 SNV in corrected 17-9-C1, and 1,121SNV in 17-14-C1 cells. Introduced correction of the CFTR exon 10 as wellas the three inserted synonymous by changes could be confirmed in bothcorrected, Cre-excised iPS cells (17-9-C1 and 17-14-C1) by whole genomesequencing. Overall there was no evidence for increased levels ofmutations in the uncorrected iPS or corrected cell lines. We found 5NSCV unique to iPS Clone 17, 2 NSCV unique to 17-9-C1, and 8 NSCV uniqueto 17-14-C1. In addition off-target ZFN binding sites, as determined asdescribed in Cradick et al. (2011) BMC Bioinformatics 12:152 were onlygenerated if each ZFN binding site was separated by 25 base pairs. Thus,there was no sequence similarity between permutations of off-target ZFNbinding sites and the NSCV found in the corrected cell lines

Cre-mediated excision of the pgk-puroTK cassette was achieved viatransient delivery of a Cre-recombinase expression plasmid. Successfulexcision of the pgk-puroTK cassette was expected to result in aphenotypic conversion of clones from puro^(R) to puro^(S) and fromFIAU^(S) to FIAU^(R) (FIG. 2).

From this process we were able to identify numerous Cre-excised clonesfrom each of the four successfully edited clones (17-1, 17-9, 17-14,17-16), and confirmed successful excision via PCR analysis and Cla Idigestion (FIG. 4; Cre-excised clones are denoted by −C1 or −C2). Asshown in FIG. 4, RT-PCR and sequencing analysis of Cre-excised clonesshowed approximately equal levels of CFTR mRNA expression from both thecorrected and mutant alleles.

Having demonstrated expression of the corrected CFTR gene allele incorrected CF iPS cells, we next examined whether we could observeup-regulation of expression of the corrected CFTR gene under in vitrodifferentiation conditions. Treatment of hES/hiPS cells with Activin Ahas previously been shown to induce the development of definitiveendoderm. See, e.g., D'Amour et al. (2005) Nat Biotechnol 23:1534-1541.

As shown in FIG. 5, the original Clone 17 CF iPS cells and corrected,Cre-excised Clone 17-9-C1 CF iPS cells, cultured in this manner for 3-5days show evidence for up-regulation of both Sox17 and CFTR mRNAs.Sequencing of the day 5 Clone 17-9-C1 CFTR RT-PCR amplicon revealedco-expression, at approximately equal levels, of both the correctedwild-type and mutant ΔF508 CFTR mRNAs.

We also cultured the Clone 17 CF iPS cells and corrected Clone 17-14 and17-16 CF iPS cells for 28 days in an air-liquid-interface (ALI)differentiation assay system previously shown to yield epithelial tissuewith certain features (e.g. cellular composition and tissuearchitecture) similar to that of lung epithelium. See, e.g., Van Hauteet al. (2009) Respir Res 10:105; Coraux et al. (2005) Am J Respir CellMol Biol 32:87-92. Under these culture conditions, CFTR expression wasupregulated. Sequencing of the day 28 Clone 17-14 and 17-16 CFTR RT-PCRamplicons revealed co-equal expression of both the corrected wild-typeand mutant ΔF508 CFTR mRNAs.

These results demonstrate appropriately regulated expression of thecorrected CFTR allele.

Example 5 Generation of Model Systems to Study CF

Thus, the compositions and methods described herein can be used togenerate model systems for the study of CF. For example, patient-derivediPSCs with corrected or disrupted ΔF508 (and/or ΔI507) provide cell andanimal models to test drugs for treatment of CF.

To mitigate concerns that phenotypes observed in downstreamcharacterization are due to variations intrinsic to the iPSC generationprocess (e.g. random integration of the reprogramming cassette),correction and disruption of ΔF508 is performed in a minimum of twoindependent iPSC lines derived from the same patient; and the sameprocess carried out on iPSCs derived from 3 unrelated patients thatcarry the mutation, thereby providing isogenic cell models for studyingΔF508 mutations in the context of different genetic backgrounds.

For certain models, the CFTR ZFNs are used to introduce DSBs to the CFTRlocus in iPSCs derived from normal subjects, and HDR invoked for de novocreation of monoallelic or biallelic ΔF508 mutations. The iPSCs arealtered as described above, except the cells are derived fromCFTR-normal subjects and the donor construct contains a nucleotidesequence that introduces the ΔF508 mutation. Clones with the expecteddigest pattern will be sequenced to verify the engineered mutation.

The impact of the ZFN-mediated gene editing on the CFTR protein and itsactivity in iPS cells is also assayed, particularly by evaluating theaccumulation of CFTR in the membrane in ZFN-modified cells as comparedto the corresponding unmodified cells and/or wild-type cells, usingimmunoblot analysis. In certain embodiments, antibodies targeted to CFTRcan provide an additional readout of CFTR activity. Furthermore,reagents can be used to detect the modification of a direct target ofCFTR. Having isogenic control cell lines adds great precision to thesemodels.

Example 6 Correction of SFTPB

The most common mutation presented in SP-B deficiency is the 121ins2(121C>GAA) mutation. Thus, ZFNs were designed to target the 121ins2(121c>GAA) SFTPB locus as described above for the CFTR locus, and usedfor gene correction.

Example 7 TALEN Design and Modification of CFTR

TALEN pairs specific for the CFTR locus are also designed, and areconstructed using both the canonical and novel RVDs as described in U.S.Publication 20110301073, incorporated by reference as described herein.TALENs are tested as described above and are active.

All patents, patent applications and publications mentioned herein arehereby incorporated by reference in their entirety.

Although disclosure has been provided in some detail by way ofillustration and example for the purposes of clarity of understanding,it will be apparent to those skilled in the art that various changes andmodifications can be practiced without departing from the spirit orscope of the disclosure. Accordingly, the foregoing descriptions andexamples should not be construed as limiting.

What is claimed is:
 1. A method of modifying a Surfactant Protein B(SP-B) gene in a cell, the method comprising; cleaving the SP-B genewith one or more zinc finger nucleases that bind to a target site in theSP-B gene.
 2. The method of claim 1, wherein the modification isselected from the group consisting of an insertion, a deletion, asubstitution and combinations thereof.
 3. The method of claim 1, furthercomprising introducing an exogenous sequence into the SP-B gene.
 4. Themethod of claim 1, wherein the modification corrects a mutation in theSP-B gene.
 5. The method of claim 4, wherein the mutation is a121 ins2mutation.
 6. A method of generating a model system for the study ofSurfactant Protein B deficiency, the method comprising modifying cellsaccording to the method of claim
 1. 7. The method of claim 6, whereinthe model system comprises a cell line.
 8. The method of claim 6,wherein the model system comprises a non-human animal.
 9. A method oftreating Surfactant Protein B deficiency in a subject, the methodcomprising modifying a SP-B gene in one or more cells of the subjectaccording to the method of claim
 4. 10. The method of claim 9, whereinthe cell is modified in vitro and the cell is administered to thesubject.